Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 February 2015 to 22 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Deviations:

yes

Remarks:

various with no effect on results or integrity of the investigation (see below)

Qualifier:

according to guideline

Guideline:

EU Method C.14 (Fish Juvenile Growth Test)

Deviations:

yes

Remarks:

various with no effect on results or integrity of the investigation (see below)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

SAMPLING
- Water samples were taken from the control and each surviving test group (cycling between replicates) for quantitative analysis.
- Samples of the fresh test preparations were taken on Days 0, 6, 13, 20 and 27.
- Samples of the expired test preparations were taken on Days 1, 7, 14, 21 and 28.
- Samples were stored frozen prior to analysis.
- Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

TEST WATER
- The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.
- Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3.
- After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 2 (attached).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST SYSTEM
- The test was carried out using juvenile rainbow trout {Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in-house since 17 February 2015.
- Fish were maintained in a glass fiber tank with a "single pass" water renewal system.
- Fish were acclimatised to test conditions from 27 February 2015 to 13 March 2015.
- The lighting cycle was controlled to give a 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- The water temperature was controlled at approximately 14 °C with a dissolved oxygen content of greater than or equal to 9.3 mg O2/L. These parameters were recorded daily.
- The stock fish were fed commercial trout pellets.
- There were three mortalities (1.1% of population) in the 7 days prior to the start of the investigation.
- On the day of test initiation, the average weight of the rainbow trout within the batch was determined to be 0.95 g, with a standard deviation of 0.13.
- The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

Not applicable

Hardness:

- Water hardness: 132 to 160 mg/L as CaCO3
- Alkalinity 78.8 to 118.8 mg/L as CaCO3

Test temperature:

14 to 16 °C

pH:

- Refer to Table 9 (attached) for details of pH measurements throughout the test.

Dissolved oxygen:

Equal or greater than 60 % of ASV (6.1 mg O2/L)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

RANGE-FINDING TEST
- Nominal loading rates of 1.0, 10 and 100 mg/L.

DEFINITIVE TEST
- Nominal loading rates of 0.30, 0.60, 1.2, 2.4 and 4.8 mg/L

Details on test conditions:

PROCEDURE
- Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

RANGE-FINDING TEST
- The loading rates to be used in the definitive test were determined by a preliminary range-finding test.
- Fish were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.
- Nominal amounts of test item (22, 220 and 2200 mg) were each separately added to the surface of 22 L of test water to give the 1.0, 10 and 100 mg/L loading rates respectively.
- After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- The stirring was stopped after 23 hours and the mixtures allowed to stand for one hour.
- A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel.
- A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. As a precautionary measure, a glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75 to 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading
rate WAFs.
- Microscopic observations of the WAFs were performed after filtering and showed no micro dispersions or undissolved test item to be present.
- In the range-finding test five fish were placed in each test and control vessel and maintained in a temperature controlled room at 15 to 16 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 14 days under semistatic test conditions.
- Each 25 to 30 L test and control vessel contained 20 L of test media and was covered to reduce evaporation.
- Any mortalities and sub-lethal effects of exposure were recorded daily from the start of exposure.
- The criteria of death was taken to be the absence of both respiratory movement and response to a physical stimulation.
- The control group was maintained under identical conditions but not exposed to the test item.

DEFINITIVE TEST
- Based on the results of the range-finding test the Sponsor requested the following loading rates were assigned to the definitive test: 0.30, 0.60, 1.2, 2.4 and 4.8 mg/L.

EXPERIMENTAL PREPARATION
- Prior to addition of the test item a glass siphon tube was placed in the test media.
- Nominal amounts of test item in duplicate (22, 26.4, 52.8 and 105.6 mg) were each separately added to the surface of 22 L of test water to give the 1.0, 1.2, 2.4 and 4.8 mg/L loading rates respectively.
- After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- The stirring was stopped after 23 hours and the mixtures allowed to stand for one hour.
- Inspection of the WAFs showed undissolved test item to be present dispersed throughout floating at the surface and at the bottom of the mixing vessel and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length).
- A length of Tygon tubing was attached to the top of the glass siphon tube, a glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75 to 100 mL
discarded) to give the 1.0, 1.2, 2.4 and 4.8 mg/L loading rate WAFs.
- Microscopic observations of the WAFs were performed after filtering and showed no micro dispersions or undissolved test item to be present. The 0.30 and 0.60 mg/L loading rate WAFs were prepared as dilutions from the 1.0 mg/L loading rate WAF preparations.

EXPOSURE CONDITIONS
- As in the range-finding test, 25-30 liter glass exposure vessels containing 20 L of test media were used for each control and test concentration.
- At the start of the test eight fish were placed in each test vessel at random, giving sixteen fish in each test loading rate.
- The test vessels were then covered to reduce evaporation and maintained at 14 to 16 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk
transition periods for a period of 28 days.
- The test vessels were aerated via narrow bore glass tubes.
- Fish were not individually identified.
- The control group was maintained under identical conditions but not exposed to the test item.
- A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products.

TEST ORGANISM OBSERVATIONS
- Any mortalities and sub-lethal effects of exposure were recorded daily throughout the test.
- The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

DETERMINATION OF FISH WEIGHT
- On Days 0, 14 and 28 the fish were anaesthetised by immersing in a solution of MS222 (tricaine
methane sulphonate), buffered with sodium carbonate.
- On Days 0 and 14, the fish were removed from the anaesthetic when locomotory movement had ceased but where respiratory movement was still evident and the weight of each fish determined and recorded.
- On Day 28, the fish were removed from the anaesthetic when locomotory movement and respiratory movement had ceased and the weight of each fish determined and recorded.

WATER QUALITY CRITERIA
- The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test.
- Measurements at 0 hours, and after each daily test media renewal, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 28 days, represent those of the used or 24-Hour old test preparations.
- The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter.
- Temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

FEEDING
- fish were fed daily during the test at a maximum level of 4% wet bodyweight with commercial trout pellets.
- The food ration was fed in two daily portions of 2 % bodyweight per portion during the week and in a single one portion of 4% bodyweight per portion at weekends.
- On days 13 and 14 the fish were only fed a single 2 % portion as the fish were anaesthetised for weighing on day 14.
- On day 20 of the test, the two portions were fed at the same time.
- The food ration fed to each exposure vessel for Days 0 to 13 was based on the mean weight of the fish on Day 0 and the food ration fed to each exposure vessel for Days 14 to 28 was based on the mean weight of the fish on Day 14.
- Excretions of the fish and any uneaten food were removed by siphon at least once per day throughout the test period.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of each mixing period.

EVALUATION OF MORTALITY DATA
- The LL50 values and associated confidence limits at 14, 21 and 28 days were calculated by Probit analysis using Linear Maximum-Likelihood regression.
- The LOEL and the NOEL at 14, 21 and 28 days were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction.
- All results were calculated using the ToxRat Professional computer software package (TOXRAT).
- An estimate of the LL50 and NOEL values at 7 days were estimated by inspection of the data.

EVALUATION OF WEIGHT DATA
- The data obtained from the measurement of fish weight on Days 0, 14 and 28 were analysed.
- The tank-average specific growth rate for each exposure vessel was calculated using the attached equation.
- Given that zero or no significant inhibition of tank-average specific growth rate was observed between the control and all the surviving test groups, it was considered inappropriate to perform regression analysis on the data and therefore the EC20 value (defined as the concentration that would diminish growth rate by 20% of its estimated size when the exposure concentration is zero) could not be calculated. However, an EC20 value was estimated from inspection of the data.
- In order to determine any significant differences between the test groups at the test start, the individual fish weight data obtained on Day 0 were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955).
- For the estimation of the Lowest Observed Effect Loading Rate (LOEL) and the No Observed Effect Loading Rate (NOEL) and in order to determine any significant differences in terms of growth between the test groups, analysis of the "pseudo" specific growth rate for individual fish on Day 28 were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955).
- All statistical analyses were performed using the SAS computer software package (SAS 1999-2001).
- The “pseudo” specific growth rate was calculated using the attached equation.
- The average r3 value for each test concentration may then be compared with the average r3 for the control enabling identification of the Lowest Observed Effect Loading Rate.

VALIDATION CRITERIA
- The results of the test are considered valid if the following criteria are met:
(i) The mortality in the control(s) should not exceed 10 per cent at the end of the test;
(ii) The mean weight of fish in the control(s) should have increased by at least half (50%) of their mean initial weight over 28 days;
(iii) The dissolved oxygen concentration must have been at least 60% of the Air Saturation Value (ASV) throughout the test;
(iv) The water temperature must not differ by more than ± 1 °C between test vessels at any one time during the test and should be maintained within a range of 2 °C within the temperature ranges specified for the test species.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOELR

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

28 d

Dose descriptor:

LOELR

Effect conc.:

2.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

RANGE-FINDING TEST
- Cumulative mortality data from the exposure of rainbow trout to the test item during the range-finding test are given in Table 1 (attached) and sub-lethal effects of exposure are given in Table 2 (attached).
- Average weight data, tank-average specific growth rates and percentage inhibition of growth of juvenile rainbow trout in the range-finding test are given in Table 3 (attached).
- By the end of the range-finding test 60 and 100% mortality had been recorded in the 10 and 100 mg/L loading rate WAF groups respectively. Sub-lethal effects (Swimming/sitting at bottom, mild hyperventilation and increased pigmentation) were observed in all test concentrations.
- Growth rates could only be determined for the control and 1.0 mg/L loading rate WAF group due to the high mortality levels recorded in the 10 and 100 mg/L loading rate WAF groups, which indicated no effect on the growth rate at a loading rate of 1.0 mg/L.
- Based on this information loading rates of 0.30, 0.60, 1.2, 2.4 and 4.8 mg/L were selected for the definitive test.
- Chemical analysis of the fresh test preparations at 0 hours (see Appendix 3, attached) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method, which was determined to be 0.13 mg/L in the 10 mg/L loading rate WAF and 0.259 mg/L in the 100 mg/L loading rate WAF.
- Chemical analysis of the old media from the 10 and 100 mg/L loading rate WAF at 24 hours showed measured test concentrations of less than the LOQ indicating that the test item was unstable under test conditions.

DEFINITIVE TEST – VERIFICATION OF TEST CONCENTRATIONS
- With the exception of the 4.8 mg/L loading rate WAF fresh test preparations on days 0 and 13 which gave measured concentration of 0.165 and 0.134 mg/L respectively, chemical analysis of the 2.4 and 4.8 mg/L loading rate WAF test preparations at all sampling occasions showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method, which was determined to be 0.13 mg/L.
- This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.
- The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

DEFINITIVE TEST – MORTALITY DATA
- Cumulative mortality data from the exposure of rainbow trout to the test item during the definitive test are given in Table 4 (attached).
- The relationship between percentage mortality and concentration at 28 days is given in Figure 1 (attached).
- Inspection of the mortality data on Days 7, 14, 21 and based on the nominal test concentrations gave the results shown in the table below.

DEFINITIVE TEST – SUB-LETHAL EFFECTS
- Sub-lethal effects of exposure were observed at 2.4 and 4.8 mg/L loading rate WAF. These responses were bullying, resulting in significant tail and fin damage (see Table 5, attached). On occasions throughout the test uneaten food was observed to be present in the 2.4 and 4.8 mg/L loading rate WAF groups.
- After 12 days of exposure 1 out of 16 and 1 out of 14 fish at 2.4 and 4.8 mg/L loading rate WAF respectively were observed to exhibit prolonged sub-lethal effects (significant tail fin damage as a result of bullying). Due to animal welfare implications (Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012) these fish were killed and classed as mortalities for the following observational time point.
- After 14 days of exposure 11 out of 12 fish at 4.8 mg/L loading rate WAF were observed to exhibit prolonged sub-lethal effects (significant tail fin damage as a result of bullying). Due to animal welfare implications (Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012) and following consultation with the Named Veterinary Surgeon, all 14 fish in the 4.8 mg/L loading rate WAF group were killed and classed as mortalities for the following observational time point.

WEIGHT DATA
- The mean weights of juvenile rainbow trout at initiation of the test and after 14 and 28 days exposure are given in Table 6 (attached) and the individual weights of juvenile rainbow trout at initiation of the test and after 14 and 28 days exposure are given in Appendix 4 (attached). The tank-average specific growth rates for the test periods 0 to 14, 14 to 28 and 0 to 28 days are given in Table 7 (attached).
- The percentage inhibition of “pseudo” specific growth rates for the test periods 0 to 14, 14 to 28 and 0 to 28 days are given in Table 8 (attached).
- The fish weight data obtained on Day 0 was compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) (see Appendix 5, attached). There were no significant differences (P>0.05) in terms of weight between the control group and all surviving test groups on Day 0.
- The control fish increased in weight by 312% of their initial mean weight over the 28 days, satisfying the validation criteria of the test that the mean weight of the fish in the control should have increased by at least half (50%) of their mean initial weight over 28 days.
- For the test periods 0 to 14, 14 to 28 and 0 to 28 days, analysis of the "pseudo” specific growth rates was compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) (see Appendix 5, attached). “Pseudo” specific growth rates were not calculated for test groups that had greater than 10% mortality. Therefore “pseudo” specific growth rate was not calculated for the 4.8 mg/L test group at 0 to 14, 14 to 28 and 0 to 28 days and the 2.4 mg/L test group at 14 to 28 and 0 to 28 days. No significant differences (P>0.05) were observed in terms of the mean "pseudo" specific growth rate between the control and each surviving test group.
- Accordingly the values shown in the table below were determined from the data. The NOEL was 1.2 mg/L as this test group showed zero mortalities, no inhibition of tank average specific growth rate, no sub-lethal effects of exposure and no significant differences (P>0.05) in terms of the “pseudo” specific growth rate when compared to the control group.

VALIDATION CRITERIA
- The test was considered to be valid given that:
(i) None of the control fish died or showed signs of stress during the test.
(ii) The mean weight of fish in the control increased by at 312 % of their mean initial weight over 28 days.
(iii) The oxygen concentration throughout the test was equal to or greater than 60 % of ASV (6.1 mg O2/L) in the control and test vessels.
(iv) The water temperature was maintained at 14 to 16 °C throughout the test.

WATER QUALITY CRITERIA
- The results of the water quality measurements are given in Table 9 (attached).
- Temperature was maintained at 14 to 16 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
- The water temperature was also recorded in the control vessel every hour using a Testo temperature logger (see Figure 2, attached).
- Water hardness and alkalinity determined at the start and end of the test for the control and the highest remaining test group (4.8 mg/L test group on days 0 and 14 and 2.4 mg/L test group on day 28) are given in Table 10 (attached). Water hardness was determined to range from 132 to 160 mg/L as CaCO3 and alkalinity was determined to range from 78.8 and 118.8 mg/L as CaCCl respectively. The test diluent also had a particulate matter content of 0.2 mg/L and a Total Organic Carbon (TOC) content of 2.07 mg/L.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of each mixing period and was observed to be a dimple at the water surface on each occasion.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- Observations on the test media were carried out during the mixing and testing of the WAFs.
- At the start of the mixing period the 1.0, 1.2, 2.4 and 4.8 mg/L loading rates were observed to be clear colourless water columns with particles of test item dispersed throughout.
- After 23 hours stirring and a one hour standing period the 1.0, 1.2, 2.4 and 4.8 mg/L loading rates were observed to remain clear colourless water columns with particles of test item dispersed throughout, floating at water surface and settled at bottom of vessel. Inspection of the WAFs showed undissolved test item to be present, therefore, the WAFs were removed by filtering through a glass wool plug (2-4 cm in length).
- After siphoning and for the duration of the test, the 1.0, 1.2, 2.4 and 4.8 mg/L loading rates were observed to be clear, colourless solutions.

Reported statistics and error estimates:

- See description of statistical methods above.

DEFINITIVE TEST MORTALITY DATA

Time (d)	LL50 (mg/L loading rate WAF)
7	> 4.8*
14	> 4.8*
21	2.8*
28	2.6*

* Not possible to calculate 95% confidence limits as data did not fit available models

DEFINITIVE TEST NOEL AND LOEL VALUES

Test period	NOEL (mg/L loading rate WAF)	LOEL (loading rate WAF)
0 to 14 days	2.4	4.8
14 to 28 days	1.2	2.4
0 to 28 days	1.2	2.4

Validity criteria fulfilled:

yes

Conclusions:

Exposure of juvenile rainbow trout to the test material for a period of 28 days resulted in no significant inhibition of the "pseudo" specific growth rate at the loading rates of 0.30, 0.60 and 1.2 mg/L over the test period of 0 to 28 days when compared to the control (zero dose) test group. Mortality rates of greater than 10% were observed at loading rates of 2.4 and 4.8 mg/L, therefore excluding these test groups from statistical analysis. The No Observed Effect Loading Rate (NOEL) was 1.2 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate (LOEL) was 2.4 mg/L loading rate WAF.

Executive summary:

GUIDELINE

A study was performed to assess the effect on growth of juvenile rainbow trout(Oncorhynchus mykiss)of the test material. The method followed the recommendations of the OECDGuidelines for Testing of Chemicals (2000) No 215, "Fish, Juvenile Growth Test” referenced asMethod C.14 of Commission Directive 2001/59/EC (which constitutes Annex V of CouncilDirective 67/548/EEC).

 

METHODS

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the

test, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, fish were exposed, in groups of 16 to a Water Accommodated Fraction (WAF) of the test item over a range of nominal loading rates of 0.30, 0.60, 1.2, 2.4 and 4.8 mg/L for a period of 28 days at a temperature of 14 to 16 °C under semi-static test conditions. Any mortalities or adverse reactions to exposure were recorded daily throughout the exposure period. The fish were fed daily at a rate of 4% bodyweight with trout pellets and the weight of each fish determined at 0, 14 and 28 days.

RESULTS

With the exception of the 4.8 mg/L loading rate WAF fresh test preparations on Days 0 and 13 which

gave measured concentration of 0.165 and 0.134 mg/L respectively, chemical analysis of the 2.4 and 4.8 mg/L loading rate WAF test preparations at all sampling occasions showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.13 mg/L). This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only. Exposure of juvenile rainbow trout to the test material for a period of 28 days resulted in no significant inhibition of the "pseudo" specific growth rate at the loading rates of 0.30, 0.60 and 1.2 mg/L over the test period of 0 to 28 days when compared to the control (zero dose) test group. Mortality rates of greater than 10% were observed at loading rates of 2.4 and 4.8 mg/L, therefore excluding these test groups from statistical analysis.

 

CONCLUSION

Exposure of juvenile rainbow trout to the test material for a period of 28 days resulted in no significant inhibition of the "pseudo" specific growth rate at the loading rates of 0.30, 0.60 and 1.2 mg/L over the test period of 0 to 28 days when compared to the control (zero dose) test group. Mortality rates of greater than 10% were observed at loading rates of 2.4 and 4.8 mg/L, therefore excluding these test groups from statistical analysis. The No Observed Effect Loading Rate (NOEL) was 1.2 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate (LOEL) was 2.4 mg/L loading rate WAF.

Reference 2

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

09 April 2015 to 04 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: HJ/T 153-2004. The guidelines for the testing of chemicals [S]. Beijing: State Environmental Protection Administration, 2004.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: CRC-MEP. The Guidelines for the Testing of Chemicals-Effects on Biotic Systems. [M]. The second edition. Beijing: China Environment Press. 2013: 135-144.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: GB/T 21806-2008, Chemicals-Fish juvenile growth test[S]. Beijing: Standardization Administration of China, 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.14 (Fish Juvenile Growth Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

SAMPLING
- The highest and lowest test concentrations were analysed when freshly prepared and immediately prior to renewal at the start of the study (0 d) and weekly thereafter (3,9, 18, 28 d).
- On each occasion, one sample collected was analysed after certain pre-treatments and the remaining samples were frozen in case further analysis was required.

Vehicle:

no

Details on test solutions:

DILUTION MEDIUM
- Good quality tap water which had been dechlorinated for at least 24 h was used.
- The total hardness of the test water was 145 mg (CaC03)/L and pH was 7.82 at room temperature.
- Characteristics of the dilution water are measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control. The latest measuring result is showed in Appendix Table 10 (attached).

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

TEST ORGANISM
- The test species of Rare minnow (Gobiocypris rarus; Batch No: JF20150115; Hatch date: 15 January 2015) were bred in house.
- A population of a single stock was used in the test.
- Details concerning the source of the fish were recorded in the raw data.
- Feeding fry were held for 14 days in holding tanks supplied with a continuous flow of aerated water before being used for testing.
- During the pre-culture, the following conditions were maintained: Light: 16 hours photoperiod daily (light intensity: 1000 lux ~ 1500 lux); temperature: 22 to 23°C; oxygen concentration: 90% of the air saturation.
--Feeding: The fish were fed daily with live food (Artemia) daily and the quantity of food was
kept constant at 4 % dry weight related to the initial fish weight (about 0.15 g).
- Food was withheld from the stock population for 24 h prior to the start of the test.
- During the holding period the tanks was inspected daily and any debris or unhealthy or dead fish
were removed.
- Fish received no treatment for disease in the two weeks preceding the test and during the test.
- After the 48-h settling-in period, no mortality was observed in the following 7 days and the batch
of fish was accepted (Batch No: JF20150115).
- Using a general anaesthetic (an aqueous solution of 100 mg/L tricaine methane sulphonate (MS 222) neutralised by the addition of two parts of sodium bicarbonate per part of MS 222), a sub-sample of 10 fish were randomly selected before the test and weighed individually as wet weights (blotted dry) with the precision of 1 mg. The wet-weight of the acclimated batch of test fish at the start of test is showed in Table 7 (attached). Handling of juvenile fish was done with the utmost care to avoid stressing and injuring test animals.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

Not applicable

Hardness:

145 mg/L as CaCO3

Test temperature:

23.1 to 23.4 °C (see Table 5, attached)

pH:

7.73 to 7.99 (see Table 3, attached)

Dissolved oxygen:

69 to 96 % of air saturation at test temperature (see Table 4, attached)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

1/5 saturated solution, 1/4 saturated solution, 1/3 saturated solution, 1/2 saturated solution and saturated solution

Details on test conditions:

APPARATUS
- Oxygen meter, Thermometer and pH meter (HACH HQ40d. The Dissolved Oxygen can be directly read by immersing the probe in the water sample when the reading is stable.)
- Equipment for determination of hardness of water (Model: 16900, made by HACH company,
the hardness can be directly read by immersing the probe in the water sample when the reading is stable.)
- Tanks made of chemically inert material, with a sealable inert lid, and with a capacity of approximately 5 L (Hai’an Sanhe Zuping Glass Instrument Factory, Jiangsu)
Analytic Balance (AG135, Accuracy 0.1 mg, METTLER TOLEDO, Switzerland; BS224S, Accuracy 0.1 mg, Sartorius, Germany);
- Thermostatic water bath (Chang Yuan Medical Instrument Factory, Jiangsu)
- UPLC-PDA (ACQUITY Ultra Performance LC, Waters USA).

TEST CONCENTRATIONS
- Based on the results of the range-finding test of fish acute toxicity, the test fish behaved normal and no death occurred in saturated solution (measured concentration, 0.284 mg/L) treatment in the 96h exposure period.
- According to the “Guidelines for the Testing of Chemicals, Effects on Biotic Systems” (the 2nd edition) (2013)”, the highest concentration for the insoluble test substance is not higher than its saturated solubility in water. Therefore the nominal concentrations of 1/5 saturated solution, 1/4 saturated solution, 1/3 saturated solution, 1/2 saturated solution and saturated solution was selected in the Juvenile Growth Test.
- Additionally, the blank control was tested in parallel (dilution medium without test substance).

PREPARATION OF TEST SOLUTIONS
- The test solution of test item was prepared by adding 1.0001 g to 1.0003 g test substance to 10 L the test medium in glass bottles with appropriate size.
- The aqueous test substance mixture was stirred for 24 hrs on a magnetic stirplate with a telfon stirbar at room temperature (about 23°C to 25°C).
- The vortex height was set at least 10% of the static liquid height.
- At the end of the 24 h, stirring was stopped and the test substance was deposited on the bottom of solution.
- The saturated solution was allowed to stand for one hour at room temperature prior to the removal of any undissolved test item by filtration through 0.45 µm millipore membrane (first approximate 100 mL discarded) to
produce a 100% v/v saturated solution of the test item.
- Details of test solutions preparation are shown in the table below.

EXPOSURE CONDITIONS
- The concentration of the test substance was demonstrated to be maintained within ± 20 % of the measured initial concentration throughout the test under a 72 h-renewal condition.
- During the test, the following conditions were maintained:
(a) Duration: 28 days.
(b) Tanks: made of chemically inert material, with a sealable inert lid, and with a capacity of approximately 5 L.
(c) Volume of the test solution: 3 L.
(d) Loading: 0.2 to 0.4 g of fish/L.
(e) Stocking densities: 10 for each treatment and control.
(f) Replicates: none.
(g) Light: 16-hour light and 8-hour dark cycle daily (light intensity: 1000 lux).
(h) Temperature: 23.1 to 23.4°C for Rare minnow.
(i) Oxygen concentration: No less than 60 per cent of the maximum air saturation value throughout the test
(j) Aeration: forbidden.
(k) Feeding: daily (quantity of food being kept constant at 4 % dry weight related to the initial fish
weight (about 0.15 g).
(l) Cleaning: remove residual fish food in the solution daily with siphon. The tank was replaced by a clean one each time the solution was renewed.

OBSERVATIONS
- Fish were examined daily during the test period and any external abnormalities (such as haemorrhage, discoloration) and abnormal behaviour (such as inactivity, strange swimming pattern, other abnormal behaviour, etc.) were noted.
- Fish were considered dead if there was no visible movement (such as gill movements) and if touching of the caudal peduncle produced no reaction. Any dead fish were removed as soon as possible.

MEASUREMENTS
- Representative samples of 10 individuals in the tank population (blotted dry) were weighed
before the test started.
- Total wet-weights (blotted dry) of fish in each concentration were also determined. At the end of the test all surviving fish were weighed as wet weights (blotted dry) by individual.
- Measurements of pH, dissolved oxygen and temperature were carried out at the beginning of the test and weekly thereafter (3, 9,18, 28 d).

VALIDITY OF TEST
- Control group: One dilution-medium control series was also run in addition to the treatment series.
- Fish loading: In the range of 0.2 to 0.4 g fish per litre.
- Validity of test result: During the whole test period, the pH values of the control mediums and test media were between 7.73 and 7.99, and the Dissolved Oxygen (DO) values varied from 69% to 96% of the air saturation at the test temperature, and the temperature of the test media was maintained at 23.1 to 23.4 °C.
- All fish in the control group were normal.
- The mean weight of fish in the controls increased more than 50%.
- The study met the acceptability criteria (dissolved oxygen concentration: no less than 60% of the air saturation value; temperature: not differ by more than ± 1 °C between test chambers; the increasing rate of the mean wet-weight of fish: no less than 50% of the initial weight).
- The test was considered valid.

STABILITY OF TEST SOLUTION AND CHEMICAL ANALYSIS
- Preparation of standard stock solution: A standard stock solution of the test substance (1000 mg/L) was prepared by dissolving 0.0501 g test substance in 50 mL acetonitrile.
- The standard working solution of 100 mg/L was prepared by diluting 5.00 mL of the above standard stock solution (1000 mg/L) to 50.0 mL with acetonitrile.
- The standard working solution of 10.0 mg/L was prepared by diluting 5.00 mL of the above standard stock solution (100 mg/L) to 50.0 mL with acetonitrile.
- Details of working solutions are given in the table below.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 0.357 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.357 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

CALIBRATION CURVE
- A series of standard solutions with concentration at 0.50, 1.00, 2.00, 5.00 and 10.0 mg/L were measured under the UPLC-PDA conditions described above.
- Based on the test result, a linear regression equation was obtained between the concentration and the UPLC-PDA response: A=1390590-55817, with good linearity of r2= 0.9988, where A represents response values, and c is the concentration of the test substance (mg/L) (see Figure 1, attached). The results show that linearity for concentration range of 0.50 mg/L to 10.0 mg/L is good.

RECOVERY TEST
- Recovery samples with concentrations of 0.10 mg/L and 1.00 mg/L were prepared by adding 0.50 mL and 5.00 mL of standard working solution (10.0 mg/L) to a total volume of 50.0 mL test water.
- Recovery water samples (20.0 mL) were extracted twice with 20.0 mL dichloromethane. The combined upper organic phases were dried with rotary evaporator, and then re-dissolved with 2.00 mL mobile phase (10-fold concentrated). The final solutions were analysed by UPLC-PDA.
- Measurements obtained from the recovery test were shown in Table 2 (attached).
- Figure 3 (attached) shows the chromatograph of the recovery sample from the recovery test.
- The mean recovery rate was 99.2% (0.10 mg/L) and 105% (1.00 mg/L). Relative standard deviation was between 4.01 % and 3.24 %.

LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION
- If the calculation is based on S/N > 3, the LOD is 0.06 mg/L.
- If the calculation is based on S/N > 10, the LOQ is 0.20 mg/L.

ANALYSIS OF TEST SUBSTANCE IN TEST SOLUTIONS
- Analytical results for the test samples are given in Table 2 (attached).
- Figure 4 and Figure 5 (attached) show spectra for the control sample and the treated sample.
- The results indicated that concentration of test substance was stable (within 80% of the initial concentration) in the water during the test period.
- Thus semi-static conditions of 72 h-renewal used in the test was acceptable.

TEST CONDITIONS
- The pH, dissolved oxygen concentration and temperature of the control and treatment groups are shown in Table 3, 4 and 5 (attached).
- During the whole test period, the pH values of the control mediums and Test Media were between 7.73 and 7.99.
- Dissolved Oxygen (DO) values varied from 69% to 96% of the air saturation at the test temperature.
- The temperature of the Test Media was maintained in 23.1 to 23.4 °C.

MORTALITY AND EFFECTS
- Mortality and visible abnormalities observed in the test fish at the observation time are shown in Table 6 (attached).
- Table 7 and Table 8 (attached) show the individual and mean fish weights on Day 0 and 28 respectively.
- No fish died in control and treatments groups. Fish in all treatments and control groups were observed alive and appeared normal.
- At the beginning of the test, fish the average weight of fish was 0.0555 g.
- Fish were chosen randomly and put into different test solutions after the temperature has been adjusted to the required value.
- At the aid of the test, the mean weight of fish in the controls increased more than 50%.
- Based on analysis of variance, the pseudo specific growth rates for each concentration were then compared with the control values using the Bartlett’s test (ANOVA, STATA 10.0). The results showed that there was no significant difference for pseudo specific growth rates in 1/5 saturated solution, 1/4 saturated solution, 1/3 saturated solution, 1/2 saturated solution and saturated solution treatments groups compared with the blank control group (p > 0.05). Therefore the LOEC for the growth rate of the test substance to rare minnow is more than saturated solution (measured concentration 0.357 mg/L), the NOEC is saturated solution (measured concentration 0.357 mg/L).

Reported statistics and error estimates:

DATA PROCESSING
- The pseudo specific growth rate (r) for the individual fish was calculated as shown in the attached equation.
- For the estimation of the lowest observed effect concentration (LOEC), and hence the no observed effect concentration (NOEC), for effects of the chemical on growth rate, the mean growth rate for each concentration were compared with the control mean using a multiple comparison method of Bartlett’s tests (ANOVA, STATA 10.0).

Validity criteria fulfilled:

yes

Conclusions:

The LOEC (lowest observed effect concentration) to juvenile rare minnow (Gobiocypris rarus) was determined to be > 0.357 mg/L and the NOEC (No observed effect concentration) of the test substance was reported as 0.357 mg/L.

Executive summary:

GUIDELINE

Under semi-static conditions of 72 h-renewal, the effect on growth rates and other observed effects in juvenile fish (Gobiocypris rarus) exposed to the test substance for 28 days was conducted according to “The guidelines for the testing of chemicals” (HJ/T 153-2004), “The Guidelines for the Testing of Chemicals, Effects on Biotic Systems” (the 2nd edition) (2013); Procedure 215 of the “Guidelines for Testing of Chemicals” of the OECD “Fish, Juvenile Growth Test” (2000) etc.

 

METHODS

Juvenile fish in exponential growth phase were placed, after being weighed, in test chambers and were exposed to a range of sub-lethal concentrations of the test substance dissolved in water under appropriate semi-static conditions. The test duration was 28 days. At the end of the test, the fish were weighed again. Effects on growth rates were analysed using a regression model in order to estimate the concentration that would cause a 50% variation in growth rate (EC50).

 

RESULTS

During the whole test period, the pH values of the control and test media were between 7.73 and 7.99, and the Dissolved Oxygen (DO) values varied from 69% to 96% of the air saturation at the test temperature. The temperature of the test media was maintained at 23.1 to 23.4°C. All fish in the control group were normal. The mean weight of fish in the control group increased more than 50%. The study met the acceptability criteria prescribed by the protocol (dissolved oxygen concentration no less than 60% of the air saturation value; temperature 23 ± 2 °C and no more than ± 1 °C between test chambers; increasing rate of the mean wet-weight of fish no less than 50% of the initial weight). The test was valid.

 

In order to confirm the stability of the test substance in the test medium, concentrations of the test samples during the test period were analysed by UPLC-PDA. Concentrations of the test substance were quantified by UPLC-PDA. As the results of the standard solution analysis, a linear regression equation was obtained with the peak area values vs. the concentration of test item (0.50, 1.00, 2.00, 5.00 and 10.0 mg/L): A=139059c-55817, with good linearity of r2= 0.9988. The result showed that linearity for the test item with the concentration range of 0.50 mg/L to 10.00 mg/L was good. The analytical results showed that the concentration of the test substance of the test solution was stable throughout a period of 72 h (deviation within 20%). Thus a semi-static test of 72 h-renewal was acceptable. The results showed that under valid semi-static test conditions (72 h-renewal), no fish died in control and treatment groups. Fish in all treatment and control groups were alive and appeared normal.

 

CONCLUSION

The LOEC (lowest observed effect concentration) to juvenile rare minnow (Gobiocypris rarus) was determined to be > 0.357 mg/L and the NOEC (No observed effect concentration) of the test substance was reported as 0.357 mg/L.

Description of key information

In the key study, involving exposure of Oncorhynchus mykiss to test item, the No Observed Effect Loading Rate (NOEL) was 1.2 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate (LOEL) was 2.4 mg/L loading rate WAF (OECD 215 and EU Method C.14). In a supporting study using Gobiocypris rarus, the LOEC (lowest observed effect concentration) to juvenile rare minnow (Gobiocypris rarus) was determined to be > 0.357 mg/L and the NOEC (No observed effect concentration) of the test substance was reported as 0.357 mg/L (GB/T 21806-2008, OECD 215, EU Method C.14).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.357 mg/L

Additional information

Key study

GUIDELINE

A study was performed to assess the effect on growth of juvenile rainbow trout (Oncorhynchus mykiss) of the test material. The method followed the recommendations of the OECD Guidelines for Testing of Chemicals (2000) No 215, "Fish, Juvenile Growth Test” referenced as Method C.14 of Commission Directive 2001/59/EC (which constitutes Annex V of CouncilDirective 67/548/EEC).

 

METHODS

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the

test, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, fish were exposed, in groups of 16 to a Water Accommodated Fraction (WAF) of the test item over a range of nominal loading rates of 0.30, 0.60, 1.2, 2.4 and 4.8 mg/L for a period of 28 days at a temperature of 14 to 16 °C under semi-static test conditions. Any mortalities or adverse reactions to exposure were recorded daily throughout the exposure period. The fish were fed daily at a rate of 4% bodyweight with trout pellets and the weight of each fish determined at 0, 14 and 28 days.

RESULTS

With the exception of the 4.8 mg/L loading rate WAF fresh test preparations on Days 0 and 13 which gave measured concentration of 0.165 and 0.134 mg/L respectively, chemical analysis of the 2.4 and 4.8 mg/L loading rate WAF test preparations at all sampling occasions showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.13 mg/L). This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only. Exposure of juvenile rainbow trout to the test material for a period of 28 days resulted in no significant inhibition of the "pseudo" specific growth rate at the loading rates of 0.30, 0.60 and 1.2 mg/L over the test period of 0 to 28 days when compared to the control (zero dose) test group. Mortality rates of greater than 10% were observed at loading rates of 2.4 and 4.8 mg/L, therefore excluding these test groups from statistical analysis.

 

CONCLUSION

Exposure of juvenile rainbow trout to the test material for a period of 28 days resulted in no significant inhibition of the "pseudo" specific growth rate at the loading rates of 0.30, 0.60 and 1.2 mg/L over the test period of 0 to 28 days when compared to the control (zero dose) test group. Mortality rates of greater than 10% were observed at loading rates of 2.4 and 4.8 mg/L, therefore excluding these test groups from statistical analysis. The No Observed Effect Loading Rate (NOEL) was 1.2 mg/L loading rate WAF. Correspondingly the Lowest Observed Effect Loading Rate (LOEL) was 2.4 mg/L loading rate WAF.

Supporting study

GUIDELINE

Under semi-static conditions of 72 h-renewal, the effect on growth rates and other observed effects in juvenile fish (Gobiocypris rarus) exposed to the test substance for 28 days was conducted according to “The guidelines for the testing of chemicals” (HJ/T 153-2004), “The Guidelines for the Testing of Chemicals, Effects on Biotic Systems” (the 2nd edition) (2013); Procedure 215 of the “Guidelines for Testing of Chemicals” of the OECD “Fish, Juvenile Growth Test” (2000) etc.

 

METHODS

Juvenile fish in exponential growth phase were placed, after being weighed, in test chambers and were exposed to a range of sub-lethal concentrations of the test substance dissolved in water under appropriate semi-static conditions. The test duration was 28 days. At the end of the test, the fish were weighed again. Effects on growth rates were analysed using a regression model in order to estimate the concentration that would cause a 50% variation in growth rate (EC50).

 

RESULTS

During the whole test period, the pH values of the control and test media were between 7.73 and 7.99, and the Dissolved Oxygen (DO) values varied from 69% to 96% of the air saturation at the test temperature. The temperature of the test media was maintained at 23.1 to 23.4°C. All fish in the control group were normal. The mean weight of fish in the control group increased more than 50%. The study met the acceptability criteria prescribed by the protocol (dissolved oxygen concentration no less than 60% of the air saturation value; temperature 23 ± 2 °C and no more than ± 1 °C between test chambers; increasing rate of the mean wet-weight of fish no less than 50% of the initial weight). The test was valid.

 

In order to confirm the stability of the test substance in the test medium, concentrations of the test samples during the test period were analysed by UPLC-PDA. Concentrations of the test substance were quantified by UPLC-PDA. As the results of the standard solution analysis, a linear regression equation was obtained with the peak area values vs. the concentration of test item (0.50, 1.00, 2.00, 5.00 and 10.0 mg/L): A=139059c-55817, with good linearity of r2= 0.9988. The result showed that linearity for the test item with the concentration range of 0.50 mg/L to 10.00 mg/L was good. The analytical results showed that the concentration of the test substance of the test solution was stable throughout a period of 72 h (deviation within 20%). Thus a semi-static test of 72 h-renewal was acceptable. The results showed that under valid semi-static test conditions (72 h-renewal), no fish died in control and treatment groups. Fish in all treatment and control groups were alive and appeared normal.

 

CONCLUSION

The LOEC (lowest observed effect concentration) to juvenile rare minnow (Gobiocypris rarus) was determined to be > 0.357 mg/L and the NOEC (No observed effect concentration) of the test substance was reported as 0.357 mg/L