Enantioselectivity in toxicity and degradation of dichlorprop-methyl in algal cultures

Enantioselectivity in the toxicity and degradation of the herbicide dichlorprop-methyl (2,4-DCPPM) in algal cultures was studied. Enantioselectivity was clearly observed in the toxicity of racemic 2,4-DCPPM and its two enantiomers. R-2,4-DCPPM showed low toxicity to Chlorella pyrenoidosa and Chlorella vulgaris, but higher toxicity to Scenedesmus obliquus. The observed toxicity was ranked: R-2,4-DCPPM>S-2,4-DCPPM>Rac-2,4-DCPPM; the toxicity of R-2,4-DCPPM was about 8-fold higher than that of Rac-2,4-DCPPM. Additionally, 2,4-DCPPM was quickly degraded, in the initial 12 h, and different algae cultures had different enantioselectivity for the 2,4-DCPPM enantiomers. There was no significant enantioselectivity for 2,4-DCPPM in Chlorella vulgaris in the initial 7 h. However, racemic 2,4-DCPPM was degraded by Scenedesmus obliquus quickly, in the initial 4 h, much quicker, in fact, than the S- or R-enantiomers (racemate>R->S-), indicating that the herbicide 2,4-DCPPM was absorbed enantioselectively by Scenedesmus obliquus. The rapid formation of 2,4-DCPP suggested that 2,4-DCPPM adsorbed by algal cells was catalytically hydrolyzed to the free acid, a toxic metabolite. The production rates of 2,4-DCPP were as follows: Scenedesmus obliquus>Chlorella pyrenoidosa>Chlorella vulgaris, consistent with the degradability of 2,4-DCPPM. Scenedesmus obliquus had quick, but different, degradative and uptake abilities for R-, S-, and Rac-2,4-DCPPM. The R- and S- enantiomers were not hydrolyzed in the first 12 h, while both enantiomers were hydrolyzed slowly after that. These results indicate that some physical and chemical properties of compounds are of importance in determining their enantioselective toxicity and degradation. The ester and its metabolite likely played an important role in enantioselective toxicity to the three algae.     

Time-dependent degradation and toxicity of diclofop-methyl in algal suspensions

Background, aim, and scope  As emerging contaminants, transformation products of the pollutants via various environmental processes are rather unknown, and some may predominately contribute to the environmental risks of the parent compounds. Hence, studies on transformation products complement the assessment of the environmental safety of the parent compounds. In this study, degradation experiments and toxicity tests using diclofop-methyl (DM), a widely used herbicide, and selected major transformation products were carried out in algal cultures to assess the time course of DM toxicity and its relevance in the formation of new breakdown products. Methods  The alga Chlorella vulgaris was maintained in the algal growth medium HB IV. The inhibition of algal growth was determined by measuring optical density at 680 nm (OD₆₈₀). Initially, DM and two selected breakdown products were added to the algal cultures, and following degradation experiments analyses were carried out by high performance liquid chromatography. In addition, the possible relationship between DM degradation and toxicity was assessed, based on physico-chemical properties of the compounds and their toxicity. Results  DM was rapidly absorbed onto the surface of the algal cells where it was hydrolyzed to diclofop (DC). Further degradation to 4-(2, 4-dichlorophenoxy) phenol (DP) occurred in the cells. However, only a minor amount of DC was degraded to DP under the same conditions when DC was initially added to the algal culture. When C. vulgaris was exposed to these compounds for 96 h, the determined EC₅₀ showed that DC was about ten times less toxic than DM (EC₅₀ = 0.42 mg/L) and that DP (EC₅₀ = 0.20 mg/L) was the most toxic. Discussion  Due to strong hydrophobicity and rare dissociation, DM has tendency toward absorption as compared to DC. The higher average degradation rates of DC initially treated by DM revealed the damage of the cell membranes caused by the DM and, thus, enhanced movement of DC into the cells. Following occurrence of phenolic breakdown products, DP suggested that DC should be intracellularly degraded to DP, which had a more potent mode of action and a higher acute toxicity. Moreover, the results for EC₅₀ at various intervals were in accordance with degradation processes of the initial compounds, in which rapid formation of DP was attributed to an increasing toxicity of DM. Conclusions  The toxicity of DM in algal suspensions increased with time due to its degradation to DP, which contributed significantly to the determined toxicity. These results indicate that the toxicity of the pesticide probably depends significantly on degradation. It is thus important to consider the time-dependent environmental processes when evaluating the toxicological effects of pesticides for proper risk assessment. Recommendations and perspectives  Increasing transformation products of these contaminants are identified in the environment, although they seem to be unknown in terms of the lacking studies on environmental behavior and ecotoxicity concerning them. Certain breakdown products probably greatly contribute to the apparent toxicity of the parent compounds, which is ascribed to the parent compounds in general studies ignoring the dependence of their toxicity on various transformation pathways. These studies that identify new intermediates and assess their toxicity via the environmental processes will be helpful to distinguish the nature of toxicity of the parent contaminants.     

Enantioselectivity of racemic metolachlor and S-metolachlor in maize seedlings

Chiral herbicides may have enantioselective effects on plants. In this study, we assessed and compared the enantioselectivity of the chiral herbicides rac-metolachlor and S-metolachlor to maize seedlings. The superoxide dismutase activity (SOD) activity of roots and stem leaves treated by rac-metolachlor was 1.38 and 1.99 times that of roots and stem leaves treated by S-metolachlor. The peroxidase activity (POD) activity of roots and stem leaves was 1.48 and 2.79 times that of roots and stem leaves treated by S-metolachlor, respectively, while the catalase activity (CAT) activity was 4.77 and 8.37 times greater, respectively. The Hill reaction activity of leaves treated by rac-metolachlor were 1.45, 1.33, and 1.14 times those treated by S-metolachlor with treatments of 18.6, 37.2, and 74.4 μM. The differences observed between treatments of rac- and S-metolachlor were significant. Significant differences in maize seedling morphology were also observed between rac- and S-metolachlor treatments. The degradation rate of S-metolachlor in roots was greater than that of rac-metolachlor. The half-lives of rac- and S-metolachlor were 80.6 and 60.3 h at 18.6 μM; 119.5 and 90 h at 37.2 μM; and 169 and 164.8 h at 74.4 μM, respectively. Using the liquid chromatography-mass spectrometry method, hydroxymetolachlor, deschlorometolachlor and deschlorometolachlor propanol were considered to be possible metabolites. We determined the enantioselective toxicity of rac- and S-metolachlor to maize and speculated on the proposed metabolic pathway of metolachlor in maize roots. These results will help to develop an understanding of the proper application of rac- and S-metolachlor in crops, and give some information for environmental safety evaluation of rac- and S-metolachlor.     

废水中2,6-二硝基酚厌氧毒性和降解动力学研究

在中温(35℃±1℃)厌氧条件下,以葡萄糖为共基质,采用间歇实验方法,研究了2,6-二硝基酚(2,6-DNP)的厌氧产甲烷毒性和厌氧降解动力学.厌氧毒性试验(ATA)以累计产甲烷量和相对活性(RA)为指标,评价了不同浓度2,6-DNP对产甲烷菌的抑制程度;结果表明,2,6-DNP浓度＜20 mg/L时,对产甲烷菌没有抑制作用,浓度为40 mg/L时产生轻度抑制,浓度为80～120 mg/L时产生重度抑制;24 h 2,6-DNP的75%、50%、25%相对抑制浓度分别为30、70和＞120 mg/L.2,6-DNP降解动力学可用Haldane方程来描述,利用非线性拟合求得动力学参数Ks、Rm、Ki分别为179.7 mg/L、4.84 mg/g VSS·h、206.5 mg/L,方差R2=0.94,拟合效果很好.     

Aquatic toxicity of cartap and cypermethrin to different life stages of Daphnia magna and Oryzias latipes

Cartap and cypermethrin, which are among the most widely used pesticides in many countries, are considered safe because of their low mammalian toxicity and their low persistence in the environment. However, recent findings of endocrine-disrupting effects and developmental neurotoxicity have raised concerns about the potential ecological impacts of these pesticides. We evaluated the aquatic toxicity of cartap [S,S′-(2-dimethylaminotrimethylene) bis(thiocarbamate), unspecified hydrochloride] and cypermethrin [(RS)-alpha-cyano-3-phenoxybenzyl-(1RS,3RS,1RS,3SR)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate], both individually and combined, on different life stages of the freshwater cladoceran Daphnia magna and a freshwater teleost, Japanese medaka (Oryzias latipes). The 96-hr Daphnia median effective concentrations (EC50s) for cartap and cypermethrin were 91.0 μ g/L and 0.00061 μ g/L, respectively. Rapid recovery of Daphnia was observed after short-term pulsed exposure to cartap and cypermethrin; there were no adverse effects on reproduction or survival 20 d after a 24 hr exposure to cartap up to 1240 μ g/L and cypermethrin up to 1.9 μ g/L. Chronic continuous exposure (for 21 d) of 7-d-old Daphnia to cypermethrin significantly reduced the intrinsic population growth rate in a concentration-dependent manner. However, because the intrinsic population growth rates were all above zero, populations did not decrease even at the highest experimental concentration of 200 ng/L. Exposure of Daphnia neonates (< 24 hr old) to cypermethrin for 21 d caused significant, sub-lethal reproduction-related problems, such as increased time to first brood, reduced brood size, and reduced total brood number, at 0.0002, 0.002, and 0.2 ng/L cypermethrin, but the intrinsic population growth rate was not significantly affected. Oryzias latipes was relatively more resistant to both pesticides. In particular, embryos appeared to be more resistant than juveniles or adults, which may be partly due to the protective role of the chorion. The incidence of larval fish deformity was significantly higher after a 96 hr exposure to as low as 250 μ g/L of cartap or 40 μ g/L of cypermethrin. The mixture of both compounds showed no synergistic toxicity. The extremely high acute-to-chronic ratio suggests that the standard acute lethal toxicity assessment might not reflect the true environmental hazards of these frequently used pesticides. Ecological hazard assessments of long-term low dose or pulsed exposures to cartap and cypermethrin may reveal more realistic consequences of these compounds in surface water.     

Acute toxicity of chlorpyrifos to embryo and larvae of banded gourami Trichogaster fasciata

This study elucidated the acute toxicity of chlorpyrifos on the early life stages of banded gourami (Trichogaster fasciata). To determine the acute effects of chlorpyrifos on their survival and development, we exposedthe embryos and two-day-old larvae to six concentrations (0, 0.01, 0.10, 1.0, 10 and 100 µg L^?1) of chlorpyrifos in plastic bowls. Log-logistic regression was used to calculate LC10 and LC50 values. Results showed that embryo mortality significantly increased with increasing chlorpyrifos concentrations. The 24-h LC10 and LC50 values (with 95% confidence limits) of chlorpyrifos for embryos were 0.89 (0.50–1.58) and 11.8 (9.12–15.4) µg L^?1, respectively. Hatching success decreased and mortality of larvae significantly increased with increasing concentrations of chlorpyrifos. The 24-h LC10 and LC50 values (with 95% confidence limits) of chlorpyrifos for larvae were 0.53 (0.27–1.06) and 21.7 (15.9–29.4) µg L^?1, respectively; the 48-h LC10 and LC50 for larvae were 0.04 (0.02–0.09) and 5.47 (3.77–7.94) µg L^?1, respectively. The results of this study suggest that 1 µg L^?1 of chlorpyrifos in the aquatic environment may adversely affect the development and the reproduction of banded gourami. Our study also suggests that banded gourami fish can serve as an ideal model species for evaluating developmental toxicity of environmental contaminants.     

Acute and chronic toxicity of organophosphate monocrotophos to Daphnia magna

The acute and chronic toxicity of monocrotophos (MCP), the binary joint toxicity of MCP and bifenthrin (BF), and sodium dodecyl benzene sulfonate (SDBS) to Daphnia magna (D. magna) was evaluated. The 24 h-median effective concentration (24 h-EC₅₀) and 48 h-median lethal concentration (48 h-LC₅₀) of MCP towards D. magna were 161 and 388 μ g/L, respectively. In addition, the lowest-observed effective concentration (LOEC) and non-observed effective concentration (NOEC) of MCP to D. magna were 10 and 5 μ g/L, respectively. Furthermore, the chronic value (ChV) of MCP against D. magna was 7 μ g/L and the acute chronic ratio (ACR) was 55. The number of offspring per female and the intrinsic rate of natural increase (r) were identified as the parameters that were most sensitive to MCP. In addition, toxic unit (TU) analysis was employed to evaluate the joint toxicities. The calculated TU_mix values of binary equitoxic mixtures of MCP + BF and MCP + SDBS were 1.47 and 1.63, respectively, which suggests that both equitoxic mixtures exert a limited antagonistic effect. The results of this study revealed that the toxic threshold of MCP towards D. magna is higher than its reported highest residue (4 μ g/L) in the ordinary aquatic environment, and that concurrent exposure to BF or SDBS may exert a slight antagonistic effect.     

A comparative study of the toxicity of 1,1,2-trichloroethane,dieldrin, pentachlorophenol and 3,4 dichloroaniline for marine and fresh water organisms

The aquatic toxicity of 1,1,2-trichloroethane, dieldrin, pentachlorophenol and 3,4-dichloroaniline was determined for about 15 marine and fresh water animals and several unicellular algae. Acute, subchronic and chronic tests including reproduction studies, were carried out. The results are summarized in five tables.     

Assessing the toxicity of organophosphorous pesticides to indigenous algae with implication for their ecotoxicological impact to aquatic ecosystems

This study aimed to determine the toxicity of three organophosphorous pesticides, chlorpyrifos, terbufos and methamidophos, to three indigenous algal species isolated from local rivers and algal mixtures. The diatom Nitzschia sp. (0.30–1.68 mg L^?1 of EC₅₀ -the estimated concentration related to a 50% growth reduction) and the cyanobacteria Oscillatoria sp. (EC₅₀ of 0.33–7.99 mg L^?1) were sensitive to single pesticide treatment and the chlorophyta Chlorella sp. was the most tolerant (EC₅₀ of 1.29–41.16 mg L^?1). In treatment with the mixture of three pesticides, Chlorella sp. became the most sensitive alga. The antagonistic joint toxic effects on three indigenous algae and algal mixtures were found for most of the two pesticide mixtures. The results suggested that mixture of pesticides might induce the detoxification mechanisms more easily than the single pesticide. The synergistic interactions between terbufos and methamidophos to algal mixtures and between methamidophos and chlorpyrifos to Nitzschia sp. indicated methamidophos might act as a potential synergist. Differential sensitivity of three families of algae to these pesticides might result in changes in the algal community structures after river water has been contaminated with different pesticides, posing great ecological risk on the structure and functioning of the aquatic ecosystem.     

Assessing the toxicity and teratogenicity of pond water in north-central minnesota to amphibians

BACKGROUND: Incidence of amphibian deformities have increased in recent years, especially in the northern region of the United States. While many factors have been proposed as being responsible for generating deformities (e.g., contaminants, ultraviolet radiation [UV], parasites), no single cause has been definitively established. METHODS: To determine whether waterborne chemicals are responsible for amphibian deformities in ponds in north-central Minnesota, we deployed semipermeable membrane devices (SPMDs) in an impacted and a reference site to accumulate lipophilic contaminants. We then exposed native tadpoles (northern leopard frogs; Rana pipiens) to the SPMD extracts combined with two agricultural pesticides (atrazine, carbaryl) at two levels of UV radiation. RESULTS AND DISCUSSION: UV radiation alone caused a slight increase in hatching success and tadpole growth rate. Deformity rate among hatchlings was high following exposure to SPMD extracts from the reference site in the absence of UV, suggesting that chemicals present at this site are broken down by UV to less harmful forms, or become less bioavailable. Conversely, impacted site SPMD extracts caused hatchling deformities only in the presence of UV, suggesting that UV potentiates the teratogenicity of the compounds present there. Impacted site SPMD extracts significantly increased the number of bony triangles among metamorphs, a common deformity observed at this site. The incidence of skin webbings increased significantly with SPMD extracts from both sites as well as with our pesticide control containing atrazine and carbaryl alone. CONCLUSIONS: Higher deformity rates among tadpoles reared in the presence of UV radiation and SPMD extracts from sites where deformities are common indicates a chemical compound (or compounds) in the water at this site may be causing the deformities. RECOMMENDATIONS AND OUTLOOK: It is important to examine the effects of chemical stressors in the presence of other natural stressors (e.g., UV radiation) to gain a better understanding of how multiple stressors work to impact amphibians and amphibian populations.     

乙酰甲胺磷对斜生栅藻的毒性及细菌降解研究

采用血球计数法测定斜生栅藻细胞数目,研究了乙酰甲胺磷对斜生栅藻的毒性作用,并通过藻类培养液中接种经过筛选得到的乙酰甲胺磷高效降解菌测定其对乙酰甲胺磷毒性的去除影响。研究结果表明,乙酰甲胺磷对斜生栅藻的抑制中浓度(EC50)大于159 mg/L,急性毒性为低毒。但进一步研究发现,在7 d后,EC50为174.68 mg/L,明显低于24 h的1 128.57 mg/L,存在亚慢性毒性。通过藻类培养液接种高效降解菌Y-6,乙酰甲胺磷对藻类的生长抑制程度减轻。     

Effects of 2,4-dichlorophenoxyacetic acid formulation on medulla spinalis of Poecilia reticulata: A histopathological study

This study investigated the possible effects of a commonly used foliar herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) formulation on medulla spinalis of lebistes. Fish were exposed to 2,4-D (15, 30, 45 mg L⁻¹), behavioral changes were monitored. Fish were fixed, histopathological examination was carried out on sections taken from the upper parts of the fish body. Histopathology showed increase in neuronal loss, swelling indicating formation of intracellular edema, vacuolization noticed as the formation of vacuoles within or adjacent to cells, deformation in the Nissl granules, pyknosis and gliosis in medulla spinalis. Behavioral changes were decreased general activity, grouping, shortness in breath, sudden rotations and jumping, loss of equilibrium and colour. In conclusion, this commercial formulation of 2,4-D is considerably neurotoxic to lebistes. Fish constitute the last link in the chain of the feeding cycle in aquatic eco-system, number of studies investigating acute and chronic neurotoxicity of various herbicides in fish should be increased.     

Water quality parameters controlling the photodegradation of two herbicides in surface waters of the Columbia Basin,Washington

The water quality parameters nitrate-nitrogen, dissolved organic carbon, and suspended solids were correlated with photodegradation rates of the herbicides atrazine and 2,4-D in samples collected from four sites in the Columbia River Basin, Washington, USA. Surface water samples were collected in May, July, and October 2010 and analyzed for the water quality parameters. Photolysis rates for the two herbicides in the surface water samples were then evaluated under a xenon arc lamp. Photolysis rates of atrazine and 2,4-D were similar with rate constants averaging 0.025 h⁻¹ for atrazine and 0.039 h⁻¹ for 2,4-D. Based on multiple regression analysis, nitrate-nitrogen was the primary predictor of photolysis for both atrazine and 2,4-D, with dissolved organic carbon also a predictor for some sites. However, at sites where suspended solids concentrations were elevated, photolysis rates of the two herbicides were controlled by the suspended solids concentration. The results of this research provide a basis for evaluating and predicting herbicide photolysis rates in shallow surface waters.     

一株芘降解菌B2的降解条件优化及降解基因

芳香烃降解菌是石油污染土壤修复的主要生物资源。采用芘平板升华法对克拉玛依原油污染土壤样品进行驯化培养,分离得到一株芘降解菌B2,经16S rDNA基因序列比对及系统发育进化分析表明,该菌株为假单胞菌属(Pseudo-monas)。采用正交设计方法优化菌株B2对高分子量多环芳烃芘的降解条件,并构建多元非线性模型预测菌株B2对芘的最佳降解条件,结果表明:在接种量OD660 nm为0.60、降解温度为40℃、降解时间为6.0 d时,预测菌株B2对芘的降解最大达到38.214 mg/L,实际测得最大降解量为37.906 mg/L,预测准确率为99.19%。运用PCR技术克隆B2的邻苯二酚-2,3-双加氧酶基因(B2C23O)(I.2.A亚家族),核酸序列分析表明,该基因全长880 bp,具有一个完整的开放阅读框,编码246个氨基酸,与已报道的Pseudomonas putida W619同源性最高为97%;对B2C23O基因编码氨基酸序列进行分析,发现其具有邻位断裂双加氧酶模式结构,推测菌株B2通过邻位裂解途径降解芘代谢中间产物邻苯二酚。     

Life cycle toxicity assessment of pesticides used in integrated and organic production of oranges in the Comunidad Valenciana, Spain

The relative impacts of 25 pesticides including acaricides, fungicides, herbicides, insecticides, and post-harvest fungicides, used in the production of oranges in Spain were assessed with current life cycle impact assessment (LCIA) tools. Chemical specific concentrations were combined with pesticide emission data and information on chemical toxicity to assess human toxicity and freshwater ecotoxicity impacts. As a case study, the relative impacts of two orange production systems in the region of Valencia, integrated pest management (IP) and organic production (OP), were assessed. The evaluation of active ingredients showed that on average acaricides have the highest human toxicity impact scores, while for freshwater ecotoxicity insecticides show the highest impact. In both impact categories the lowest impact scores were calculated for herbicides. In the production of 1 kg of orange fruits, where several kinds of pesticides are combined, results show that post-harvest fungicides can contribute more than 95% to the aggregate human toxicity impacts. More than 85% of aquatic ecotoxicity is generated by fungicides applied before harvest. The potential to reduce impacts on freshwater ecosystems is seven orders of magnitude, while impacts on human health can be reduced by two orders of magnitude. Hence, this stresses the importance of a careful pre-selection of active ingredients. In both impact categories, organic production represents the least toxic pest-control method.     

光电催化氧化法降解藻毒素MCLR

使用DSA阳极,对光电催化氧化降解藻毒素MCLR的效能及其影响因素进行了研究。结果表明,电极表面的TiO2在光催化氧化降解MCLR的过程中发挥了明显的光催化作用。在光降解、电催化氧化、光催化氧化和光电催化氧化4个过程中,光电催化氧化对MCLR和TOC的去除率最高,分别可达100%和13%,并且光电催化氧化的去除率大于光催化氧化和电催化氧化之和,表明后两者的耦合过程产生了一定的协同作用。辐照光源和电流密度存在最佳匹配条件,分别为UVC辐照、电流密度10 mA/cm2和UVA辐照、电流密度1.0 mA/cm2,此条件下光电协同作用最显著。在光电催化氧化过程中,随极板间距增大而出现的去除率下降取决于电催化过程,而不是光催化过程;光电催化氧化MCLR的去除率随其初始浓度增加而减小。     

Molecular identification and nanoremediation of microbial contaminants in algal systems using untreated wastewater

Wastewater-algal biomass is a promising option to biofuel production. However, microbial contaminants constitute a substantial barrier to algal biofuel yield. A series of algal strains, Nannochloris oculata and Chlorella vulgaris samples (n = 30), were purchased from the University of Texas, and were used for both stock flask cultures and flat-panel vertical bioreactors. A number of media were used for isolation and differentiation of potential contaminants according to laboratory standards (CLSI). Conventional PCR amplification was performed followed by 16S rDNA sequencing to identify isolates at the species level. Nanotherapeutics involving a nanomicellar combination of natural chitosan and zinc oxide (CZNPs) were tested against the microbial lytic groups through Minimum Inhibitory Concentration (MIC) tests and Transmission Electronic Microscopy (TEM). Results indicated the presence of Pseudomonas spp., Bacillus pumilus/ safensis, Cellulosimicrobium cellulans, Micrococcus luteus and Staphylococcus epidermidis strains at a substantial level in the wastewater-fed algal reactors. TEM confirmed the effectiveness of CZNPs on the lytic group while the average MICs (mg/mL) detected for the strains, Pseudomonas spp, Micrococcus luteus, and Bacillus pumilus were 0.417, 3.33, and 1.458, respectively. Conclusively, CZNP antimicrobials proved to be effective as inhibitory agents against currently identified lytic microbial group, did not impact algae cells, and shows promise for in situ interventions.     

Identifying the cause of toxicity in an algal whole effluent toxicity study - an unanticipated toxicant

Toxicity was observed in whole effluent toxicity (WET) studies with the freshwater alga, Pseudokirchneriella subcapitata, in three consecutive monthly studies, (NOEC = 50-75%). Toxicity was not observed to Ceriodaphnia dubia or the fathead minnow, Pimephales promelas in concurrent studies. Selected toxicity identification evaluation (TIE) tests were conducted in a tiered approach to eliminate possible toxicants and progressively identify the causative agent. Filtration following alkaline adjustment (pH 10 or 11) was effective in eliminating significant growth effects and also reduced phosphate concentration. The TIE studies confirmed that the observed effluent toxicity was caused by excess ortho-phosphate in the effluent not by overstimulation or related to unfavorable N:P ratios; but due to direct toxicity. The 96-h 25% inhibition concentration (IC25) of ortho-phosphate to P. subcapitata was 3.4 mg L⁻¹ while the maximum acceptable toxicant concentration was 4.8 mg L⁻¹. This study illustrates the value of multi-species testing and also provides an example of an effective TIE using algae identifying an unanticipated toxicant.