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Long-term toxicity to fish

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Administrative data

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: HJ/T 153-2004, The guidelines for the testing of chemicals, SEPA, 2004

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The Guidelines for the Testing of Chemicals, Effects on Biotic Systems CRC-MEP (2nd edition, 2013)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: GB/T 21806-2008, Chemicals- Fish juvenile growth test, Beijing, SAC (2008)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

- Purity: >99%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken (at least in duplicate) from each freshly prepared (0 d) concentration and old medium (3, 9, 28 d). On each occasion, one sample collected was analysed by UPLC-PDA; the remaining samples were retained in case further analysis would be required.

Test solutions

Vehicle:

no

Details on test solutions:

- Preparation of standard stock solution: A standard stock solution of the test substance I (1108 mg/L) was prepared by dissolving 0.0277 g test substance into 50.0 mL acetonitrile. The standard stock solution II of 100 mg/L was prepared by drawing 9.00 mL above standard stock solution I (1108 mg/L) and bringing to 100 mL with acetonitrile.
- Test solution: The test solutions were prepared by directly dissolving appropriate amount of test substance in dilution medium and then facilitating its dispersion by stirring for 2 hours. The test solutions were renewed every 3 days.

Test organisms

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

TEST ORGANISM
- Common name: Rare minnow
- Strain: Gobiocypris rarus
- Source: Key Lab of Pesticide Environmental Assessment and Pollution Control, Nanjing Institute of Environmental Sciences, MEP, 8 Jiang-wang-miao Street, Nanjing 210042, China
- Age at study initiation: Juvenile
- Weight at study initiation: Mean weight was 0.0633 to 0.0639 g (RSD: 0-1.73%)
- Feeding during test: Twice daily; Quantity of food being kept constant at 4% dry weight related to the initial fish weight.
- Food type: Live food (Artemia)

ACCLIMATION
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: 1000-1500 lux)
-Temperature: 22°C 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 being kept constant at 4% dry weight related to the initial fish weight (about 0.15g). Food was withheld from the stock population for 24 h prior to the start of the test.
-During the holding period the tanks were inspected daily and any debris or unhealthy or dead fish were removed.

Study design

Test type:

semi-static

Water media type:

other: Good quality tap water which had been dechlorinated for at least 24 h

Limit test:

no

Total exposure duration:

28 d

Test conditions

Test temperature:

22.8-23.1°C

pH:

7.65-7.91

Dissolved oxygen:

83.0-94.0% of air saturation

Nominal and measured concentrations:

Nominal concentrations: 1.00, 2.00, 4.00, 5.00, 8.00, 10.0 mg/L
Measured concentrations: 1.08, 1.92, 4.10, 8.05 9.95 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Chemically inert tank, with a sealable inert lid
- Size of vessel: 5 L
- Volume of test solution: 3 L
- Aeration: Not less than 60 per cent of the maximum air saturation value throughout the test.
- Renewal rate of test solution: 72 hour
- Loading: 0.1 to 0.6 g of fish/L
- Stocking densities: 10 for each treatment and control
- Replicates: none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Good quality tap water which had been dechlorinated for at least 24 h was used
- Total organic carbon: 1.62 mg/L
- Total hardness: 185 mg CaCO3/L
- Intervals of water quality measurement: Twice a year

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light and 8-hour dark cycle daily
- Light intensity: 1000 Lux

EFFECT PARAMETERS MEASURED:
- Observations: The 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 (e.g. gill movements) and if touching of the caudal peduncle produces no reaction. The dead fish were removed as soon as possible.

- Measurements: All individuals in the tank population (blotted dry) were weighed before the test started. The 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. Each freshly prepared treatment (day 0) and each old test medium (day 3, 9, 28) were analysed. Measurements of pH, dissolved oxygen and temperature were carried out at the beginning and thereafter (3, 9, 28 d).

TEST CONCENTRATIONS
Based on the results of the Acute Toxicity to rare minnow, 96 h-LC50 was 36.0 mg/L (based on measured concentration), 96 h-LC0 was 10.0 mg/L (measured concentration 9.90 mg/L). Therefore 1.00, 2.00, 4.00, 8.00 and 10.0 mg/L were selected in the Juvenile Growth Test. In addition, the blank control was tested in parallel (dilution medium without test substance).

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

4.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Effect concentrations 2

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 4.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Effect concentrations 3

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

4.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

behaviour

Effect concentrations 4

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

8.05 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

behaviour

Details on results:

It was found that fish in the 1.00, 2.00, 4.00 mg/L treatment and control groups were observed alive and appeared to have normal. Some of fish were observed abnormal effect (lying on side or back) and were dead at concentrations of 8.00 mg/L and 10.0 mg/L. At the beginning of the test, the average weight of the control fish was is 0.0636 g, and 0.1425 g at the end of the test. At the end of the test, the mean weight of the control fish increased more than 50%. The results showed that there was no significant difference for pseudo specific growth rates in 1.00, 2.00, 4.00 mg/L treatments groups compared with the control group (p >0.05). The mortality rates of 8.00 and 10.0 mg/L treatment exceed 10%, and therefore data from these treatments was not used to develop pseudo specific growth rates for the analysis of growth.

Reported statistics and error estimates:

Based on analysis of variance, the pseudo specific growth rates for each concentration were compared with the control values using the Bartlett's test.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

28 d-LOEC (weight): > 4.00 mg/L (measured concentration 4.10 mg/L)
28 d-NOEC (weight): 4.00 mg/L (measured concentration 4.10 mg/L)
28 d-LOEC (behaviour): 8.00 mg/L (measured concentration 8.05 mg/L)
28 d-NOEC (behaviour): 4.00 mg/L (measured concentration 4.10 mg/L)

Executive summary:

Under semi-static conditions of 72 h-renewal, the effect on growth rate and other observed effects in juvenile fish (Gobiocypris rarus) exposed to the test substance for 28 days was conducted.

During the whole test period, the pH values of the control and test media were between 7.65 and 7.91, and the Dissolved Oxygen (DO) values varied from 83.0% to 94.0% of the air saturation at the test temperature, and the temperature of the test media was maintained at 22.8°C~23.1°C.

All fish in the control group were normal. The mean weight of fish in the control group increased more than 50%. So 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 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). Therefore, 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 with principal peaks which are considered representative of reaction products making up the complex nature. As the results of the standard solution analysis, a linear regression equation was obtained with the peak area values vs. the concentration of the test substance (0.00, 1.00, 2.00, 4.00, 5.00, 8.00, 10.0 mg/L): A=10552C+554.46, with good linearity of r2 = 0.9995. The result showed that linearity for the test substance with the concentration range of 0.00 mg/L to 10.0 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 reasonable.

It was found that fish in 1.00, 2.00, 4.00 mg/L treatments and control groups were observed alive and appeared normal. Some of fish were observed to have abnormal effects (lying on side or back) and dead at concentrations of 8.00 mg/L and 10.0 mg/L treatments.

The results showed that under valid semi-static test conditions (72 h-renewal), the LOEC (the lowest observed effect concentration) and NOEC (the no observed effect concentration) for weight (growth) and the LOEC and the NOEC for behaviour were as follows:

28 d-LOEC (weight): >4.00 mg/L (measured concentration 4.10 mg/L)

28 d-NOEC (weight): 4.00 mg/L (measured concentration 4.10 mg/L)

28 d-LOEC (behaviour): 8.00 mg/L (measured concentration 8.05 mg/L)

28 d-NOEC (behaviour): 4.00 mg/L (measured concentration 4.10 mg/L)