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Long-term toxicity to fish

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Reference
Endpoint:
fish, juvenile growth test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 March 2015 to 09 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 215 (Fish, Juvenile Growth Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: HJ/T 153-2004, The guidelines for the testing of chemicals [S]. Beijing: SEPA, 2004
Deviations:
no
Qualifier:
according to
Guideline:
other: CRC-MEP. The Guidelines for the Testing of Chemicals, Effects on Biotoc Systems [M]. 2nd edition. Beijing: China Environment Press. 2013: 135-144
Deviations:
no
Qualifier:
according to
Guideline:
other: GB/T 21806-2008, Chemicals- Fish juvenile growth test, Beijing: SAC, 2008
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Water samples (20.0 mL) were taken (at least in duplicate) from each concentration during the definitive test at 0, 24, 48, 72 and 96 hours.
- The remaining samples were retained to permit further analysis if necessary.
Vehicle:
no
Details on test solutions:
DILUTION MEDIUM
- Good quality tap water, which had been dechlorinated for at least 24 hours was used.
- The total hardness of the test water was 145 mg CaCO3/L and pH was 7.82 at room temperature.
- Characteristics of the dilution water were measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control and the most recent result is shown in Table 10 (attached).
Test organisms (species):
other: Gobiocypris rarus
Details on test organisms:
- Fish (batch number: JF20150115; hatch date: 15 January 2015) were bred in-house.
- A population of a single stock was used in the test and details were recorded in the raw data.
- Feeding fry were kept for 14 days in holding tanks supplied with a continuous flow of aerated water before being used for testing.
- During pre-culture a 16-hour photoperiod (light intensity 1000 to 1500 lux), oxygen concentration of 90 % air saturation and a temperature of 22 to 23 °C was maintained.
- The fish were fed twice daily with live food (Artemia) and the quantity of food was kept constant in relation to initial fish weight at 4 % dry weight (approximately 0.15 g). Food was withheld from the stock population for 24 hours prior to the start of the test.
- During the holding period the tanks were inspected daily and any debris or unhealthy/dead fish were removed. Fish received no treatment for disease in the two weeks preceding the test or during the test.
- After a 48-hour acclimation period, no mortality was observed in the following 7 days and the batch of fish was accepted.
- 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 subsample of 10 fish were blotted dry and weighed individually before the test. The resulting wet weight of the acclimated batch of test fish at the start of the test is shown 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 mgL as CaCO3
Test temperature:
23.1 to 23.5 °C (see Table 5, attached)
pH:
7.84 to 8.04 (see Table 3, attached)
Dissolved oxygen:
74 to 94 % of air saturation (see Table 4, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
0 %, 1 %, 2 %, 4 %, 5 %, 8 % and 10 % v/v saturated solution
Details on test conditions:
APPARATUS
- Oxygen meter, thermometer and pH meter (HACH HQ40d)
- Hardness of water (HACH Model 16900).
- 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 (METTLER TOLEDO AG135, Switzerland, accuracy 0.1 mg; Sartorius BS224S, Germany, accuracy 0.1 mg).
- Thermostatic water bath (Chang Yuan Medical Instrument Factory, Jiangsu).
- ICP-MS (Thermo Scientific X Series 2, USA).

PREPARATION OF TEST SOLUTIONS
- The test solution was prepared by adding 0.3000 to 0.3003 g of test item to 3 L of test medium in glass bottles of an appropriate size.
- The aqueous test substance mixture was stirred for 24 hours on a magnetic stirplate with a Teflon stirbar at room temperature (25 °C). The vortex height was set at at least 10 % of the static liquid height.
- After 24 hours, stirring was stopped and the test substance was allowed to float on the water.
- The stock solution was settled for one hour at room temperature prior to removal of any undissolved test item by filtration through a 0.45 µm Millipore membrane (first approximate 100 mL discarded) to produce a 100 % v/v stock solution of the test item.
- Details of the test solutions prepared from the 100 % v/v stock solution are shown in the table below.

CONDITIONS OF EXPOSURE
- The concentration of test substance was demonstrated to be within ± 20 % of the measured initial concentration throughout the test under a 72-hour renewal condition.
- Tanks were made of chemically inert material with a sealable inert lid and with a capacity of approximately 5 L.
- Volume of test solution was 3 L.
- Loading was 0.2 to 0.4 g of fish/L.
- Stocking density was 10 fish for each treatment group and the control.
- No replicates were used.
- Photoperiod was 16 hours light and 8 hours dark (intensity 1000 lux).
- Temperature was 23.1 to 23.5 °C.
- Oxygen concentration was not less than 60 % of the maximum air saturation value throughout the test and aeration was forbidden.
- Fish were fed daily with the quantity of food being kept constant with respect to initial fish weight at 4 % dry weight (approximately 0.15 g).
- Residual fish food in the solution was removed daily with a siphon. The tank was replaced each time the solution was renewed.

OBSERVATIONS
- Fish were examined daily during the test period and any external abnormalities (such as haemorrhage or discolouration) and abnormal behaviour (such as inactivity or abnormal swimming pattern) were noted.
- Fish were considered dead if there was no visible gill movement and touching of the caudal peduncle produced no reaction. Dead fish were removed from the tank as soon as possible.

MEASUREMENTS
- Representative samples of 10 individuals in the tank population were blotted dry and weighed before the start of the test.
- The total wet weight of fish (blotted dry) at each test concentration was also determined.
- All fish (blotted dry) were weighed individually at the end of the test.
- Since all fish at the highest treatment concentration (10 % v/v saturated stock solution) died, the second highest concentration were analysed at 0 days and weekly thereafter (3, 9, 18 and 28 days).
- Measurements of pH, dissolved oxygen and temperature were performed at the beginning of the test and weekly thereafter (3, 9, 18 and 28 days).

VALIDITY OF THE TEST
- One dilution-medium control series was run in addition to the treatment series.
- Fish loading was in the range 0.2 to 0.4 g fish/L.
- During the whole test period, the pH values of the control and test solutions were between 7.87 and 8.04, the dissolved oxygen concentration varied from 74 % to 94 % of air saturation and temperature was maintained at 23.1 to 23.5 °C.
- All fish in the control group were normal.
- The mean weight of fish in the control group increased by more than 50 %.
- The study met acceptability criteria (dissolved oxygen concentration no less than 60 % of air saturation; temperature difference not more than ± 1 °C between test chambers; the increasing rate of fish mean wet weight no less than 50 % of initial weight).

STABILITY OF TEST SOLUTION AND CHEMICAL ANALYSIS
- A standard stock solution (I) of the test substance (89.0 mg/L) was prepared by dissolving 0.0089 g of test item in 100 mL methanol.
- A standard solution (II) of 10.0 mg/L v/v was prepared by adding 5.618 mL of standard stock solution I (89.0 mg/L) to 50.0 mL of 4 % nitric acid solution.
- A standard stock solution (III) of 1.00 mg/L v/v was prepared by adding 5.00 mL of standard stock solution II (100 mg/L) to 50.0 mL of 4 % nitric acid solution.
- Working solutions were prepared by pipetting appropriate amounts of standard solution into 10.0 mL of 4 % nitric acid solution. Details of the working solutions are shown in the table below.

ICP-MS CONDITIONS
- Main operating parameters are shown in the table below.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.171 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 0.171 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
ANALYTICAL METHOD FOR DETERMINATION OF TEST SUBSTANCE IN WATER
(1) CALIBRATION CURVE
- A series of standard solutions with concentrations of 0.02, 0.05, 0.10, 0.20 and 0.30 mg/L were measured using the ICP-MS conditions described.
- Based on the test result, a linear regression equation of A = 867816c + 6030.3 was obtained from the concentration and ICP-MS response where A represents response values of boron and c is the concentration of test substance (mg/L) (see Figure 1, attached).
- Results showed that linearity was good for the concentration range of 0.02 to 0.30 mg/L (r2 = 0.993).
- Recovery samples with concentrations of 0.10 mg/L were prepared by adding 5.00 mL of standard stock solution III (1.00 mg/L) to a total volume of 50.0 mL test water.
- Recovery samples with concentrations of 1.00 mg/L were prepared by adding 5.00 mL of standard stock solution II (10.0 mg/L) to a total volume of 50.0 mL test water.
- Recovery water samples of 1.00 mg/L (5.00 mL) were diluted to 50.0 mL v/v with 4 % nitric acid solution and then analysed by ICP-MS. The recovery water samples of 0.10 mg/L were directly analysed by ICP-MS.
- Measurements obtained from the recovery test are shown in Table 2 (attached).
- The chromatograph of the recovery sample from the recovery test is shown in Figure 3 (attached).
- The mean recovery rates were 96.3 % (1.00 mg/L) and 97.1 % (10.0 mg/L).
- Standard deviations were 2.72 % (1.00 mg/L) and 2.38 % (10.0 mg/L).
(2) DETECTION LIMIT
- The minimum detection limit for test item using ICP-MS was determined to be 1.0 x 10E-10 g.
- The minimum quantitation detection concentration for water samples was 0.01 mg/L.

ANALYSIS OF TEST SUBSTANCE IN TEST SOLUTIONS
- The analysed results for the test samples are given in Table 2 (attached).
- Spectra for the control sample and the treated sample are shown in Figures 4 and 5 (attached).
- The results indicated that concentration of test substance was stable (within 80 % of initial concentration) in the water during the test period.
- Semi-static conditions involving 72-hour renewal were therefore considered reasonable.

TEST CONDITIONS
- The pH dissolved oxygen concentration and temperature of the control and treatment groups during the test are shown in Tables 3, 4 and 5 (attached).
- During the whole test period, the pH values of the control and test solutions were between 7.87 and 8.04 and the dissolved oxygen concentrations varied from 74 to 94 % of the air saturation. The temperature of the solutions was maintained at 23.1 to 23.5 °C.

MORTALITY AND EFFECTS
- Mortality and visible abnormalities observed in test fish are shown in Table 6 (attached).
- Individual and mean fish weights on Day 0 and Day 28 are shown in Tables 7 and 8 (attached).
- The results showed that, under valid semi-static test conditions with 72-hour renewal, no fish died in the control or 1 %, 2 %, 4 % or 5 % stock solution treatment groups.
- Effects (fish lying on side or back) occurred at nominal test concentrations of 8 % and 10 % stock solution.
- All fish were dead after 3 days exposure at nominal test concentration of 10 % stock solution.
- The results were slightly different from an acute toxicity test suggesting that the juveniles may be more sensitive to the test substance.
- At the beginning of the test, the average weight of fish in the control group was 0.0574 g and this value increased to 0.124 g at the end of the test. The mean weight of fish in the control group therefore increased by more than 50 %.
- Based on analysis of variance, the pseudo specific growth rates for each concentration were then compared with the control values using Bartlett’s test (ANOVA, STATA 10.0). The results (see Table 9, attached) showed that there were no significant difference for pseudo specific growth rates in 1 %, 2 %, 4 %, 5 % and 8 % stock solution treatment groups compared with the blank control group (p > 0.05).
- The LOEC for growth rate was therefore > 8 % stock solution (measured concentration 0.171 mg/L) and the NOEC was 8 % stock solution (measured concentration 0.171 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 estimation of the Lowest Observed Effect Concentration (LOEC) and hence the No Observed Effect Concentration (NOEC), the mean growth rate for each concentration was compared with the control mean using Bartlett's multiple comparison method (ANOVA, STATA 10.0).
Validity criteria fulfilled:
yes
Conclusions:
The LOEC (28 d) was determined to be > 8 % v/v saturated solution (measured concentration 0.171 mg/L) and the NOEC (28 d) was reported as 8 % v/v saturated solution (measured concentration 0.171 mg/L).
Executive summary:

GUIDELINE

The test was designed to determine the effect on growth rates and other endpoints in juvenile fish (Gobiocypris rarus) exposed to test substance. The study met the requirements of HJ/T 153-2004, The guidelines for the testingof chemicals [S], Beijing: SEPA, 2004, CRC-MEP; The Guidelines for the Testing of Chemicals, Effects on Biotic Systems [M] 2nd edition, Beijing: China Environment Press, 2013: 135-144;GB/T21806-2008,Chemicals - Fish juvenile growth test, Beijing: SAC, 2008; Guidelines for Testing of Chemicals, 215,Fish, Juvenile Growth Test [S], OECD,2000.

 

METHODS

In accordance with results from an acute toxicity test, fish were exposed to nominal concentrations of 1 %, 2 %, 4 %, 5 %, 8 % and 10 % v/v saturated solution prepared by diluting a saturated (100 %) stock solution (0.3000 to 0.3003 g test substance dissolved in 3 L test media). Concentrations of the test substance were quantified by ICP-MS. A linear regression equation A = 867816c + 6030.3 was obtained from the peak area values versus the concentration of test item (0.02, 0.05, 0.10, 0.20 and 0.30 mg/L) and good linearity was observed (r2 = 0.993). The analytical results showed that mean concentration of test item with nominal concentrations of 1 % and 8 % v/v saturated solutionwere 0.020 mg/L and 0.171 mg/L respectively. The test solution was stable throughout the 72 hour investigation (deviation within 20 %) showing that a semi-static test with 72-hour renewal was reasonable.

 

RESULTS

The pH values of the control and test media were between 7.84 and 8.04 during the test. Dissolved oxygen values varied from 74 % to 94 % of air saturation and the temperature of test media was maintained at 23.1 to 23.5 °C. The mean weight of the fish in the control group increased by > 50 %. The study met the acceptability criteria of the protocol (dissolved oxygen concentration no less than 60 % of air saturation; temperature 23 ± 2 °C with a difference of no more than ± 1 °C between chambers; the increasing rate of fish mean wet weight no less than 50 % of initial weight). Under the valid, semi-static, test conditions, no fish died in the control or 1 %, 2 %, 4 % and 5 % v/v saturated solution treatment groups. Effects (fish lying on side or back) occurred at nominal test concentrations of 8 % and 10 % v/v saturated solution. All fish were dead after three days exposure at a nominal concentration of 10 % v/v saturated solution. This result was slightly different to the acute toxicity result and suggested the juveniles may be more sensitive to the test substance.

 

CONCLUSION

The LOEC (28 d) was determined to be > 8 % v/v saturated solution (measured concentration 0.171 mg/L) and the NOEC (28 d) was reported as 8 % v/v saturated solution (measured concentration 0.171 mg/L).

Description of key information

The LOEC (28 d) was determined to be > 8 % v/v saturated solution (measured concentration 0.171 mg/L) and the NOEC (28 d) was reported as 8 % v/v saturated solution (measured concentration 0.171 mg/L).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.171 mg/L

Additional information

GUIDELINE

The test was designed to determine the effect on growth rates and other endpoints in juvenile fish (Gobiocypris rarus) exposed to test substance. The study met the requirements of HJ/T 153-2004, The guidelines for the testingof chemicals[S],Beijing: SEPA, 2004, CRC-MEP; The Guidelines for the Testing of Chemicals, Effects on Biotic Systems[M]2nd edition,Beijing:China Environment Press, 2013: 135-144;GB/T21806-2008,Chemicals - Fishjuvenilegrowth test, Beijing: SAC, 2008; Guidelines for Testing of Chemicals, 215,Fish, Juvenile Growth Test[S],OECD,2000.

 

METHODS

In accordance with results from an acute toxicity test, fish were exposed to nominal concentrations of 1 %, 2 %, 4 %, 5 %, 8 % and 10 % v/v saturated solution prepared by diluting a saturated (100 %) stock solution (0.3000 to 0.3003 g test substance dissolved in 3 L test media). Concentrations of the test substance were quantified by ICP-MS. A linear regression equation A = 867816c + 6030.3 was obtained from the peak area values versus the concentration of test item (0.02, 0.05, 0.10, 0.20 and 0.30 mg/L) and good linearity was observed (r2 = 0.993). The analytical results showed that mean concentration of test item with nominal concentrations of 1 % and 8 % v/v saturated solution were 0.020 mg/L and 0.171 mg/L respectively. The test solution was stable throughout the 72 hour investigation (deviation within 20 %) showing that a semi-static test with 72-hour renewal was reasonable.

 

RESULTS

The pH values of the control and test media were between 7.84 and 8.04 during the test. Dissolved oxygen values varied from 74 % to 94 % of air saturation and the temperature of test media was maintained at 23.1 to 23.5 °C. The mean weight of the fish in the control group increased by > 50 %. The study met the acceptability criteria of the protocol (dissolved oxygen concentration no less than 60 % of air saturation; temperature 23 ± 2 °C with a difference of no more than ± 1 °C between chambers; the increasing rate of fish mean wet weight no less than 50 % of initial weight). Under the valid, semi-static, test conditions, no fish died in the control or 1 %, 2 %, 4 % and 5 % v/v saturated solution treatment groups. Effects (fish lying on side or back) occurred at nominal test concentrations of 8 % and 10 % v/v saturated solution. All fish were dead after three days exposure at a nominal concentration of 10 % v/v saturated solution. This result was slightly different to the acute toxicity result and suggested the juveniles may be more sensitive to the test substance.

 

CONCLUSION

The LOEC (28 d) was determined to be > 8 % v/v saturated solution (measured concentration 0.171 mg/L) and the NOEC (28 d) was reported as 8 % v/v saturated solution (measured concentration 0.171 mg/L).