Phenol, styrenated

Endpoint summary

Description of key information

Short term toxicity to fish:

Study was designed to assess the effect of test chemical on the mortality of fish. Test conducted for 96 hrs. The lethal concentration (LC50) value of test chemical in fish in a 96 hr study on the basis of mortality effect was determine to be 5.6 mg/L. The test chemical is highly insloluble in water the obtained LC50 value is much higher than the solubility of test chemical. Thus, by considering the solubility, test chemical cannot be toxic at its solubility limit.

 

Long term toxicity to fish:

A fish sexual development test (FSDT) was performed with zebrafish (Danio rerio). The objective of this study was the assessment of effects of continuous exposure to 4-(1 -phenylethyl)-phenol (4-MSP) on the early life stages and sexual differentiation of zebrafish (Danio rerio), following the OECD test guideline 234. The study was conducted with nominal concentrations of 2.0, 6.3, 20.0, 63.2 and 200 μg/L in four replicates each under flow through conditions. An untreated control was run in parallel. Exposure was started with 30 fertilised eggs per test vessel and replicate. Endpoints that were determined and which were not indicative for endocrine-mediated effects included hatching success and rates, and mortalities during the early life stage and the juvenile growth. At day 35 post fertilization (pf) and when groups were terminated (day 63 pf), fish were digitally photographed. Fish lengths were determined by evaluating photographs using electronically supported analysis. Single wet weights (blotted dry) were determined on day 63 pf (test end). An endpoint that is indicative for endocrine-mediated effects and was determined during the study was the sex ratio. Sex ratios were determined macroscopically by inspection of the gonads and by histopathological verification. Blood samples of all fish were taken and measured for the vitellogenin (VTG) concentration, a biomarker also indicative for endocrine modes of action. Furthermore, a histopathological examination of the fish gonads was performed.

 

Chemical analysis

The concentrations of the 4-MSP were assessed by chemical analysis using GC-MS. The LOQ was set to 0.75 μg 4-MSP/L. Mean concentrations per treatment of the 4-MSP during the course of the study were between 92.0 % and 105.7 % of the nominal concentration of the test item. As some samples differed from the desired 80 – 120 % of the nominal values, it was decided to base the biological effects on mean measured concentrations (2.1 μg/L; 6.4 μg/L; 19.7 μg/L; 61.8 μg/L;
 187.9 μg/L).

 

Non-endocrine apical endpoints:
 Early life stage

· Hatching rate: Hatch was completed at 5 to 6 dpf in all replicates, with no difference between treatments.

· Post-hatch survival at 35 dpf: There was no test-item related effect on post-hatch survival at 35 dpf when compared to controls. 

· Length at 35 dpf: There was no test-item related effect on length at 35 dpf when compared to controls.

 

Non-endocrine apical endpoints: Test termination

· Post-hatch survival at 63 dpf: There was no test-item related effect on post-hatch survival at 63 dpf when compared to controls.

· Length and weight at 63 dpf: There was no test-item related effect on length and weight at 63 dpf when compared to controls. No differences in terms of length and weight at test end were observed for males and females, respectively. Of note, for males, no values could be obtained for the highest treatment replicates, as no mature male fish could be identified (details in section “Sex ratio” below).

 

Endocrine related endpoints and biomarkers: Sex ratio (based on histopathology)

When compared to controls, a statistically significant test-item related effect on sex ratio in terms of %males was reported at 187.9 μg/L. Indeed, no mature males were found in any of the replicates of the highest test concentration whereas the mean value of %males ranged between 18.6 % and 30.0 % in controls and the four lower test concentrations. The NOEC for the endpoint sex ratio (%males) was thus defined as 61.8 μg/L (mean measured concentration).

The mean value of %females/stage 0 (individuals where the gonads showed only undeveloped ovaries with oogonia to perinucleolar oocytes) ranged between 8.9 % (controls) and 52.5 % (187.9 μg/L), with a monotonous concentration- response relationship. A statistically significant difference was observed for concentrations ≥ 6.4 μg/L.

Regarding the number of females/stage 0, the NOEC was defined as 2.1 μg/L (mean measured concentration).

 

Endocrine related endpoints and biomarkers: Vitellogenin content in blood plasma

When compared to controls, a statistically significant test-item related increase in vitellogenin content was reported at 187.9 μg/L in females only (mean VTG value of 729.7 ng/μg protein vs. 480.8 ng/μg protein in controls).

VTG contents for males could be determined only for controls and for the four lower test concentrations, as no mature males were present at the highest test level. No significant differences in VTG content were observed.

Based on the results for mature females, the NOEC for the biomarker VTG was determined at 61.8 μg /L (mean measured concentration).

Short term toxicity to aquatic invertebrates:

Aim of this first study was to evaluate the effect of test chemical on the mobility of aquatic invertevbrates. Test conducted for 48 hrs. The effective concentration (EC50) value of test chemical in aquatic invertebrate (Daphnia sps.) in a 48 hr study on the basis of mobility effect was observed to be 4.6 mg/L. The test chemical is highly insloluble in water the obatined LC50 value is much higher than the solubility of test chemical. Thus, by considering the solubility, test chemical cannot be toxic at its solubility limit.

Long term toxicity of aquatic invertebrate:

Aim of this study was to determine the long term effect of test chemical on the aquatic invertebrate daphnia magna for 21 days. After the exposure of test chemical with daphnia magna, effect were observed and calculated. The EC50 value after 21 days of exposure were determine to be 1.5 mg/l and NOEC was at 0.2 mg/l.

Toxicity to aquatic algae and cyanobacteria:

The study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). The test solution was prepared in aseptic condition. As the test substance has a very low water solubility value, a stock WAF (Water Accommodated Fraction) solution was prepared by adding 100 mg of test substance in 1 Litre of BBM with a constant mechanical stirring for 48 hours. The remaining test solutions were prepared by dilution from the stock WAF. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate. After 72 hours of exposure to test item to various nominal test concentrations, EL50 was determine to be 20.4213 mg/l graphically and and EL30 was observed to be at 7.943 mg/l

Toxicity to microorganisms:

Toxicity of test chemical were checked by observing effect of test chemical on the oxygen consumption of activated sludge. Test conducted for 3 hrs. The effective concentration (EC50) value of test chemical on microorganism (activated sludge) in a 3 hr study was determine to be 362 mg/L.

Additional information

Summarized study results for the toxicity of test chemical on the growth and other behavior of test organisms fish, invertebrates, algae, cyanobacteria and microorganisms. The studies are as mentioned below:

Short term toxicity to fish:

Data available for the test chemical from various sources, study has been reviewed to determine the short term toxicity on fish of the test chemical. The studies are as mentioned below:

Study was designed to assess the effect of test chemical on the mortality of fish. Test conducted for 96 hrs. The lethal concentration (LC50) value of test chemical in fish in a 96 hr study on the basis of mortality effect was determine to be 5.6 mg/L. The test chemical is highly insloluble in water the obtained LC50 value is much higher than the solubility of test chemical. Thus, by considering the solubility, test chemical cannot be toxic at its solubility limit.

 

Similar short term toxicity of test chemical were studied on the mortality of fish. The lethal concentration LC0, LC50 and LC 100 value of test chemical in fish (Brachydanio rerio) in a 96 hr study on mortality effect was determine to be 1, 3.2 and 10 mg/L. The test chemical is highly insloluble in water the obtained LC50 value is much higher than the solubility of test chemical. Thus, by considering the solubility, test chemical cannot be toxic at its solubility limit.

Thus based on the above data available from various sources, chemical consider to be non toxic.

Long term toxicity to fish:

Various experimental studies of the test chemical were reviewed for long term toxicity to fish endpoint which are summarized as below:

 

In an experimental key study, a fish sexual development test (FSDT) was performed with zebrafish (Danio rerio). The objective of this study was the assessment of effects of continuous exposure to 4-(1 -phenylethyl)-phenol (4-MSP) on the early life stages and sexual differentiation of zebrafish (Danio rerio), following the OECD test guideline 234. The study was conducted with nominal concentrations of 2.0, 6.3, 20.0, 63.2 and 200 μg/L in four replicates each under flow through conditions. An untreated control was run in parallel. Exposure was started with 30 fertilised eggs per test vessel and replicate. Endpoints that were determined and which were not indicative for endocrine-mediated effects included hatching success and rates, and mortalities during the early life stage and the juvenile growth. At day 35 post fertilization (pf) and when groups were terminated (day 63 pf), fish were digitally photographed. Fish lengths were determined by evaluating photographs using electronically supported analysis. Single wet weights (blotted dry) were determined on day 63 pf (test end). An endpoint that is indicative for endocrine-mediated effects and was determined during the study was the sex ratio. Sex ratios were determined macroscopically by inspection of the gonads and by histopathological verification. Blood samples of all fish were taken and measured for the vitellogenin (VTG) concentration, a biomarker also indicative for endocrine modes of action. Furthermore, a histopathological examination of the fish gonads was performed.

 

Chemical analysis

The concentrations of the 4-MSP were assessed by chemical analysis using GC-MS. The LOQ was set to 0.75 μg 4-MSP/L. Mean concentrations per treatment of the 4-MSP during the course of the study were between 92.0 % and 105.7 % of the nominal concentration of the test item. As some samples differed from the desired 80 – 120 % of the nominal values, it was decided to base the biological effects on mean measured concentrations (2.1 μg/L; 6.4 μg/L; 19.7 μg/L; 61.8 μg/L;
 187.9 μg/L).

 

Non-endocrine apical endpoints:
 Early life stage

· Hatching rate: Hatch was completed at 5 to 6 dpf in all replicates, with no difference between treatments.

· Post-hatch survival at 35 dpf: There was no test-item related effect on post-hatch survival at 35 dpf when compared to controls. 

· Length at 35 dpf: There was no test-item related effect on length at 35 dpf when compared to controls.

 

Non-endocrine apical endpoints: Test termination

· Post-hatch survival at 63 dpf: There was no test-item related effect on post-hatch survival at 63 dpf when compared to controls.

· Length and weight at 63 dpf: There was no test-item related effect on length and weight at 63 dpf when compared to controls. No differences in terms of length and weight at test end were observed for males and females, respectively. Of note, for males, no values could be obtained for the highest treatment replicates, as no mature male fish could be identified (details in section “Sex ratio” below).

 

Endocrine related endpoints and biomarkers: Sex ratio (based on histopathology)

When compared to controls, a statistically significant test-item related effect on sex ratio in terms of %males was reported at 187.9 μg/L. Indeed, no mature males were found in any of the replicates of the highest test concentration whereas the mean value of %males ranged between 18.6 % and 30.0 % in controls and the four lower test concentrations. The NOEC for the endpoint sex ratio (%males) was thus defined as 61.8 μg/L (mean measured concentration).

The mean value of %females/stage 0 (individuals where the gonads showed only undeveloped ovaries with oogonia to perinucleolar oocytes) ranged between 8.9 % (controls) and 52.5 % (187.9 μg/L), with a monotonous concentration- response relationship. A statistically significant difference was observed for concentrations ≥ 6.4 μg/L.

Regarding the number of females/stage 0, the NOEC was defined as 2.1 μg/L (mean measured concentration).

 

Endocrine related endpoints and biomarkers: Vitellogenin content in blood plasma

When compared to controls, a statistically significant test-item related increase in vitellogenin content was reported at 187.9 μg/L in females only (mean VTG value of 729.7 ng/μg protein vs. 480.8 ng/μg protein in controls).

VTG contents for males could be determined only for controls and for the four lower test concentrations, as no mature males were present at the highest test level. No significant differences in VTG content were observed.

Based on the results for mature females, the NOEC for the biomarker VTG was determined at 61.8 μg /L (mean measured concentration).

 

In a supporting study, a range finder pre-test was performed prior to a fish sexual development test (FSDT) with zebrafish (Danio rerio). The range finder pre-test design was based on the OECD 210 Fish Early-Life Stage (FELS) test, but with a reduced test setup consisting of 3 concentrations plus a control, in two replicates each. The tested concentrations were 0, 0.02, 0.2 and 2.0 mg/L, and the following parameters were monitored: hatch, post-hatch survival and growth. Hatch was found to be delayed in the highest test concentration. Hatch mainly occurred on day 4 post fertilization in the lower dose groups, but only on day 5 in the highest dose group. Mortality started to occur in the highest dose group at day 6 -7 post fertilization. At study termination, all larvae were found dead. At the two other treatment levels, the post-hatch survival rate was similar as that of the control. At study termination, no effect on the growth or dry weight of the surviving larvae of the lower dose groups was observed.At 2.0 mg/L (nominal), 100% mortality was observed. At lower dose levels (0.2 and 0.02 mg/L, nominal), no effects on mortality or growth were observed.On the basis of effect on mortality of the test organism, the 21 d NOEC value was determined to be 0.2 mg/l.

 

On the basis of the above results of long term toxicity to fish study as per OECD TG 234 of 4-(1 -phenylethyl)-phenol (4-MSP), the 21 d NOEC value was determined to be in the range of 0.0618 to > 0.18 mg/l, respectively. Based on the OECD TG 210 study of 2,4,6-tris(1-phenylethyl)phenol, the 21 d NOEC value was determined to be 0.2 mg/l.

 

Short term toxicity to aquatic invertebrates:

Data available for test chemicals including structurally and functionally similar read across chemical, study has been reviewed to determine the short term toxicity of the test chemical on the growth and mobility of daphnia magna. The studies are as mentioned below:

 

Aim of this study was to evaluate the effect of test chemical on the mobility of aquatic invertevbrate. Test conducted for 48 hrs. The effective concentration (EC50) value of test chemical in aquatic invertebrate (Daphnia magna) in a 48 hr study on the basis of mobility effect was observed to be 4.6 mg/L. The test chemical is highly insloluble in water the obatined EC50 value is much higher than the solubility of test chemical. Thus, by considering the solubility, test chemical cannot be toxic at its solubility limit.

 

Above study was supported by the second data from peer reviewed journal. Short term toxicity of test chemical were studied was observing the effect of test chemical on the intoxication of freshwater aquatic invertebrate. 10 neonates <24h old Daphnia pulex in each test chamber not fed during the test were used in the study. Test conducted under the static system for 48 hrs. Based on the intoxication of Daphnia pulex after the exposure of 48 hrs, the EC50 was observed to 1.44 mg/l. The test chemical is highly insloluble in water the obatined EC50 value is much higher than the solubility of test chemical. Thus, by considering the solubility, test chemical cannot be toxic at its solubility limit.

 

Thus based on the above results, chemical consider to be non toxic at its maximum solubility limit.

 

Long term toxicity to aquatic invertebrate:

Aim of this study was to determine the long term effect of test chemical on the aquatic invertebrate daphnia magna for 21 days. After the exposure of test chemical with daphnia magna, effect were observed and calculated. The EC50 value after 21 days of exposure were determine to be 1.5 mg/l and NOEC was at 0.2 mg/l.

 

Toxicity to aquatic algae and cyanobacteria:

Summarized result for the toxicity of test chemical on the growth of algae were mention below:

 

The study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). The test solution was prepared in aseptic condition. As the test substance has a very low water solubility value, a stock WAF (Water Accommodated Fraction) solution was prepared by adding 100 mg of test substance in 1 Litre of BBM with a constant mechanical stirring for 48 hours. The remaining test solutions were prepared by dilution from the stock WAF. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate. After 72 hours of exposure to test item to various nominal test concentrations, EL50 was determine to be 20.4213 mg/l graphically and and EL30 was observed to be at 7.943 mg/l

 

Above study was supported by second study from authoritative database. Study was conducted to determine the effect of test chemical on the growth of algae. The no observed effect concentration (NOEC) value of test chemical in aquatic algae in a 72 hr study on the basis of growth rate effect and areas under the growth curves was determine to be 3.2 and 1.8 mg/L respectively. Also the EC50 value was reported to be 9.7 mg/L.

 

 

Toxicity to microorganisms:

Toxicity of test chemical were checked by observing effect of test chemical on the oxygen consumption of activated sludge. Test conducted for 3 hrs. The effective concentration (EC50) value of test chemical on microorganism (activated sludge) in a 3 hr study was determine to be 362 mg/L.