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Long-term toxicity to fish

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Reference
Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Remarks:
The long-term fish toxicity study was conducted solely to comply with a non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was conducted under a National quality assurance scheme (HJ/T 155-2004) similar to OECD GLP
Qualifier:
according to guideline
Guideline:
other: HJ/T 153-2004, The guidelines for the testing of chemicals [S] Beijing SEPA 2004
Qualifier:
according to guideline
Guideline:
other: CRC-MEP. The Guidelines for the Testing of Chemicals, Effects on Biotoc Systems [M] 2nd Edition Beijing: China Environment Press 2013 113-120
Version / remarks:
2nd edition
Qualifier:
equivalent or similar to guideline
Guideline:
other: GB/T 21807-2008, Chemicals-Fish acute toxicity test, Beijing SAC 2008
Qualifier:
according to guideline
Guideline:
OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)
Qualifier:
according to guideline
Guideline:
EU Method C.15 (Fish, Short-term Toxicity Test on Embryo and Sac-fry Stages)
GLP compliance:
no
Remarks:
The study was conducted under a National quality assurance scheme (HJ/T 155-2004) similar to OECD GLP
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations (1, 2, 4, 8 and 10 mg/L)
- Sampling method: 10 mL water samples were taken from the middle of each aquarium, in duplicate, at the start of the test, and days 3, 9 and 11 days in fresh and aged solutions
- Sample storage conditions before analysis: Not reported
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 1000 mg/L stock solution was prepared by adding 1.00 g test item in 1 L. The test solution was diluted by the stock solution.
- Controls: Dilution water control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported
Test organisms (species):
other: Gobiocypris rarus
Details on test organisms:
TEST ORGANISM
- Common name: Rare minnow
- Age at study initiation (mean and range, SD): Fertilised eggs were taken at random and examined under microscope to confirm the batch of eggs were in the same cell stage at the start of the test.
- Method of breeding: Male and female fish were paired at 1:1 ration in an incubator. When the male was observed chasing the female, eggs and sperm were collected from the fish and the eggs artificially inseminated to get fertilised eggs.
- Feeding during test: None

ACCLIMATION
- Acclimation period: None
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
11 d
Remarks on exposure duration:
72 hour media reneweal
Post exposure observation period:
None
Hardness:
184.9 mg/L as CaCO3
Test temperature:
25.0 °C
pH:
7.63 - 7.96
Dissolved oxygen:
70 - 97 %
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
Nominal: 1.00, 2.00, 4.00, 8.00 and 10.0 mg/L
Mean measured: 0.87, 1.94, 3.89, 7.90 and 10.1 mg/L
Details on test conditions:
TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): Not reported
- Test vessel: 1 L test chambers with sealable inert lids
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass tanks containing 1L test media
- Aeration: Not reported
- Renewal rate of test solution (frequency/flow rate): 72 hour renewal
- No. of fertilized eggs/embryos per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Biomass loading rate: 10 eggs/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water, dechlorinated for at least 24 hours
- Total organic carbon: 1.62 mg/L
- Particulate matter: Not reported
- Metals: As = < 0.001 mg/L, Co = 0.0001 mg/L, Pb = < 0.001 mg/L, Co = < 0.001 mg/L, Ni = < 0.001 mg/L, Hg = < 0.0002 mg/L, Cu = < 0.01 mg/L, Cr(VI) = < 0.004 mg/L, Fe = < 0.01, Zn = 0.055 mg/L and Ag < 0.001 mg/L
- Pesticides: Malthion = < 0.025 mg/L, methyl parathion = < 0.002 mg/L, parathion = < 0.003 mg/L
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Salinity: Not applicable
- Culture medium different from test medium: No
- Intervals of water quality measurement: Twice per year

OTHER TEST CONDITIONS
- Adjustment of pH: None reported
- Photoperiod: 16-hour light and 8-hour dark cycle
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Stage of embryonic development at test start; hatching and survival of eggs, embryos, larvae and juvenile fish, abnormal appearance and abnormal behaviour at 0, 1, 2, 3, 4, 5, 6, 7 8, 9, 10 and 11 days; and length at the end of the test.
Reference substance (positive control):
no
Key result
Duration:
11 d
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Key result
Duration:
11 d
Dose descriptor:
EC50
Effect conc.:
> 10.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Key result
Duration:
11 d
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival
Key result
Duration:
11 d
Dose descriptor:
EC50
Effect conc.:
> 10.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Survival
Key result
Duration:
11 d
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: malformations
Key result
Duration:
11 d
Dose descriptor:
EC50
Effect conc.:
> 10.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: malformations
Key result
Duration:
11 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: hatching, survival and malformations
Key result
Duration:
11 d
Dose descriptor:
NOEC
Effect conc.:
10.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: hatching, survival and malformations
Key result
Duration:
11 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: hatching, survival and malformations
Key result
Duration:
11 d
Dose descriptor:
LOEC
Effect conc.:
> 10.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: hatching, survival and malformations
Key result
Duration:
11 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
11 d
Dose descriptor:
NOEC
Effect conc.:
10.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
11 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
11 d
Dose descriptor:
LOEC
Effect conc.:
> 10.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Details on results:
- Mortality/survival at embryo and larval stages:
- Overall mortality/survival: Survival rates ranged between 96.6 % and 100 %
- Days to hatch and numbers hatched: Hatching rates ranged between 93.3 % and 100 %
- Data for length and weight of surviving fish: There was no significant difference between test solution treatment groups and the control group for fish length (Bartlett's test, p > 0.05)
- Type of and number with morphological abnormalities: No abnormal morphology observed in any treatment groups or the control
- Type of and number with behavioural abnormalities: No abnormal behaviour observed in any treatment groups or the control
- Other biological observations: None reported
- Effect concentrations exceeding solubility of substance in test medium: No
- Incidents in the course of the test which might have influenced the results: None
Results with reference substance (positive control):
No positive control
Reported statistics and error estimates:
The hatching, survival and abnormal rates were adjusted by the rates in the control group by Abbott's formula.
Trimmed Spearman-Karber Method was used to calculate the EC50 and confidence limits.
The NOEC and LOEC for fish length growth was compared to the control using a multiple comparison method of Bartlett's test (p > 0.05)

Hatching and Survival Observed in the Test Fish:

Time (d) Nominal concentration (mg/L)
Blank control 1.00 2.00 4.00 8.00 10.0
Hatching Survival1 Hatching Survival Hatching Survival Hatching Survival Hatching Survival Hatching Survival
0 0 30 0 30 0 30 0 30 0 30 0 30
1 0 30 0 29 0 30 0 30 0 29 0 30
2 0 30 0 29 0 29 0 30 0 28 0 30
31 10 29 9 29 13 29 11 30 10 28 14 30
4 29 29 28 28 29 29 30 30 28 28 29 29
5 29 29 28 28 29 29 30 30 28 28 29 29
6 29 29 28 28 29 29 30 30 28 28 29 29
7 29 29 28 28 29 29 30 30 28 28 29 29
8 29 29 28 28 29 29 30 30 28 28 29 29
9 29 29 28 28 29 29 30 30 28 28 29 29
10 29 29 28 28 29 29 30 29 28 28 29 28
112 29 28 28 28 29 28 30 29 28 27 29 28
Hatching rates (%) 96.7 93.3 96.7 100 93.3 96.7 96.7 93.3 96.7 100 93.3 96.7
Survival rates (%)3 100 100 100 96.7 100 96.6 100 100 100 96.7 100 96.6

1: Initiation of hatching on the 3rd day

2: The larvae of the control group begin to die of hunger on the 11th day, and the test ended. The hatching rates and survival rates were calculated based on the results of the first 10 days.

3: Survival rates = (survival number/hatching number)*100 %

Abnormal behaviour and abnormal appearence of test fish:

Time (d) Nominal concentration (mg/L)
Blank control 1.00 2.00 4.00 8.00 10.0
0 30NB 30NB 30NB 30NB 30NB 30NB
1 30NB  29NB & 1 Dead 30NB 30NB 29NB & 1 Dead 30NB
2 30NB 29NB & 1 Dead 29NB & 1 Dead 30NB 28NB & 2 Dead 30NB
31 29NB & 1 Dead 29NB & 1 Dead 30NB 30NB 28NB & 2 Dead 30NB
4 29NB & 1 Dead 28NB & 2 Dead 29NB & 1 Dead 30NB 28NB & 2 Dead 29NB & 1 Dead
5 29NB & 1 Dead 28NB & 2 Dead 29NB & 1 Dead 30NB 28NB & 2 Dead 29NB & 1 Dead
6 29NB & 1 Dead 28NB & 2 Dead 29NB & 1 Dead 30NB 28NB & 2 Dead 29NB & 1 Dead
7 29NB & 1 Dead 28NB & 2 Dead 29NB & 1 Dead 30NB 28NB & 2 Dead 29NB & 1 Dead
8 29NB & 1 Dead 28NB & 2 Dead 29NB & 1 Dead 30NB 28NB & 2 Dead 29NB & 1 Dead
9 29NB & 1 Dead 28NB & 2 Dead 29NB & 1 Dead 29NB & 1 Dead 28NB & 2 Dead 29NB & 1 Dead
10 29NB & 1 Dead 28NB & 2 Dead 29NB & 1 Dead 29NB & 1 Dead 28NB & 2 Dead 28NB & 2 Dead
112 28NB & 2 Dead 28NB & 2 Dead 28NB & 2 Dead 29NB & 1 Dead 27NB & 3 Dead 28NB & 2 Dead

NB: normal appearance and behaviour

1: Initiation of hatching on the 3rd day

2: The larvae of the control group begin to die of hunger on the 11th day, and the test ended.

Hatching rate, Survival rate, and normal rate of the Embryos and Larvae at the End of the Test (11 days)

Nominal Concentration (mg/L) Measured concentration (mg/L) Adjusted hatching rate, survival rate and normal rate (%)*
Hatching rate Survival rate Normal rate
Blank Control ND 100 100 100
1.00 0.87 96.5 100 100
2.00 1.94 100 100 100
4.00 3.89 103.4 96.7 100
8.00 7.9 96.5 100 100
10.0 10.1 100 96.6 100
EC50 (mg/L, based on measured concentration) > 10.1 > 10.1 > 10.1
95 % CI (mg/L, based on measured concentration) - - -
NOEC (mg/L) 10.1
LOEC (mg/L) > 10.1

*Adjusted by Abbot's formula, Adjusted hatching rate, survival rate and normal rate were calculated based on the results of day 10

Validity criteria fulfilled:
yes
Remarks:
Dissolved oxygen concentration >60% air saturation value, temperature not differing by more than 1.5°C between test chambers, survival of fertilised eggs in control >80% and post-hatch success in controls >90%.
Conclusions:
Based on hatching, survival and malformation the EC50 was >10.1 mg/L, the overall NOEC was 10.1 mg/L and the overall LOEC was >10.1 mg/L, based on mean measured concentrations.
Executive summary:

The toxicity of the test item to the rare minnow (Gobiocypris rarus) was determined in an 11-day semi-static GLP study according to OECD TG 212. Fertilised eggs were taken at random and examined under microscope to confirm the batch of eggs were in the same cell stage at the start of the test. The fertilised eggs were exposed to nominal concentrations of 1.00, 2.00, 4.00, 8.00 and 10.0 mg/L (mean measured: 0.87, 1.94, 3.89, 7.9 and 10.1 mg/L), and a blank water control. The test medium was exchanged after 72 hours.

Based on hatching, survival and malformation the EC50 was >10.1 mg/L, the overall NOEC was 10.1 mg/L and the overall LOEC was >10.1 mg/L, based on mean measured concentrations.

Description of key information

11-day NOEC = 10.1 mg/L, rare minnow (Gobiocypris rarus), OECD TG 212, Guixiang 2016

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
10.1 mg/L

Additional information

The toxicity of the test item to the rare minnow (Gobiocypris rarus) was determined in an 11-day semi-static GLP study according to OECD TG 212. Fertilised eggs were taken at random and examined under microscope to confirm the batch of eggs were in the same cell stage at the start of the test. The fertilised eggs were exposed to nominal concentrations of 1.00, 2.00, 4.00, 8.00 and 10.0 mg/L (mean measured: 0.87, 1.94, 3.89, 7.9 and 10.1 mg/L), and a blank water control. The test medium was exchanged after 72 hours.

Based on hatching, survival and malformation the EC50 was >10.1 mg/L, the overall NOEC was 10.1 mg/L and the overall LOEC was >10.1 mg/L, based on mean measured concentrations.