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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Nov 2015 - 10 Dec 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.15 (Fish, Short-term Toxicity Test on Embryo and Sac-fry Stages)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SEPA 2004, HJ/T 153-2004, The guidelines for the testing of chemicals
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: CRC-MEP, The Guidelines for the Testing of Chemicals, Effects on Biotic Systems
Version / remarks:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: GB/T 21807-2008, Chemicals-Fish acute toxicity test
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and all test substance treatments
- Sampling method: 10.0 mL water samples were taken at the middle of the aquarium (in duplicate) at the beginning of the test (0 d), after 3, 9 and 13 days in fresh and aged medium. One sample was analysed, the remaining sample was retained in case further analysis would be required.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Slow stir stock solution. The test solution (100 mg/L) was prepared by adding 0.3001 - 0.3007 g test substance in 3 L dilution water. The aqueous test substance mixture was stirred for 24 hrs on a magnetic stirplate and a teflon stirbar at 30 °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 settled down to the bottom. The stock solution stands for 1 hour at room temperature prior to the removal of any undissolved test item by filtration through 0.45 um millipore membrane (first approximate 100 mL discarded) to produce the stock solution of the test item. The other concentrations were prepared from the stock solutions.
- Controls: Blank control (no test item)
Test organisms (species):
other: Gobiocypris rarus (rare minnow)
Details on test organisms:
TEST ORGANISM
- Common name: Gobiocypris rarus (rare minnow)
- Source: Commerical fish supplier of Institute of Hydrobiology, Chinese Academy of Sciences.
- Method of breeding: Parent individuals were healthy adult fish without surface damage from a breeding population in a recirculating system.
- Feeding during test : no
- Food type 14 d before test: brine shrimp, twice daily.

BREEDING
- The male and female fish were paired 1:1 ratio in incubator. When the male was observed chasing the female, eggs and sperm were collected from the fish and the eggs artificially inseminated to get fertilized eggs. Before the test, the fertilized eggs were taken at random under a microscope to confirm the batch of eggs were in the same cell stage (picture available in report).
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
12 d
Hardness:
184.9 mg CaCO3/L
Test temperature:
24.8 - 25.0 °C
pH:
7.55 - 7.83
Dissolved oxygen:
86 - 99% of the air saturation at the test temperature.
Nominal and measured concentrations:
Nominal: 10, 20, 40, 80 and 100% stock solution (prepared at 100 mg/L)
Mean measured: 18.5, 34.6, 78.0, 145 and 196 ug/L. See Table 1 in 'Any other information on results' for details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 L glass tank with a sealable inert lid
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 24 h renewal
- No. of fertilized eggs/embryos per vessel: 10 eggs/L
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Good quality tap water which had been dechlorinated for at least 24 h. The total hardness of the test water was 184.9 mg CaCO3/L and pH was 8.17 at room temperature. Characteristics of the dilution water are measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control.
- Total organic carbon: 1.62 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH, dissolved oxygen and temperature were analysed at start of the study (0 d), in fresh and aged medium after 3, 9 and 12 d.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light and 8 h dark cycle daily.
- Light intensity: 1000 - 1500 lux

EFFECT PARAMETERS MEASURED:
- Stage of embryonic development: at start of exposure to verify embyonic stage (blastocyst stage) as precisely as possible. This was done using a representative sample of eggs suitably preserved and cleared.
- Hatching and survival: at least once daily. Dead embryos, larvae and juvenile fish were removed as soon as observed.
- Abnormal appearance: number of larvae showing abnormality of body form was recorded and the nature of the abnormality described.
- Abnormal behaviour: e.g. hyperventilation, uncoordinated swimming, atypical quiescence and atypical feeding behaviour were recorded during the test.
- Length: At the end of the test, total length was measured. A general anaesthetic MS 222 was used to make fish go into coma without pain, and then the fish were put into the alcohol after ensuring the fish will not wake up again.
Reference substance (positive control):
no
Key result
Duration:
12 d
Dose descriptor:
NOEC
Effect conc.:
196 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Number hatched, mortality, morphology and length
Remarks on result:
other: This is considered to be the maximum solubility of the test substance in test medium.
Details on results:
- Overall survival in controls and treatments: 93.0 - 100%
- Hatching rate in controls and treatments: 93.3 - 100%
- Days to hatch: 3 days
- Data for length of surviving fish: 6.62 - 6.89 mm mean total length; no significant difference between treatment groups and control.
- Type of and number with morphological abnormalities: none observed
- Type of and number with behavioural abnormalities: none observed
- Other biological observations: larvae in the control group began to die from hunger at 12 d and the test ended.

EC50 (hatching, survival, malformation): >100% stock solution (mean measured concentration 196 ug/L)
LOEC (hatching, survival, malformations, length): > 100% stock solution (mean measured concentration 196 ug/L)
Reported statistics and error estimates:
EC5- values: Trimmed Spearman-Karber method (Version 1.5, USEPA)
LOEC and NOEC: Multiple comparison method Bartlett's test (ANOVA, STATA 10.0)

 Table 1: Stability Test Results of Test Substance in Test Media

Nominal Concentration

Analysed Concentration (ug/L)

Mean #

Day 0

Day 3

Day 9

Day 12

Fresh

Aged

Fresh

Aged

Fresh

Aged

Blank control

ND *

ND

ND

ND

ND

ND

ND

10% stock solution

18.5

20.4

19.3

19.7

18.2

17.0

16.6

20% stock solution

34.6

37.5

38.3

31.9

34.8

33.3

32.1

40% stock solution

78.0

85.5

75.5

73.9

79.8

76.5

76.8

80% stock solution

145

159

139

137

147

143

142

100% stock solution

196

215

187

188

200

195

190

#: The average of the anlysed concentrations of fresh and aged test solutions on days 0, 3, 9 and 12. The concentration value in this table has considered the dilution factor.

* ND: not detected.

Table 2: Hatching and survival Observed in the Test Fish 

(a) Initiation of hatching on the third day; (b) The larvae in the control group began to die from hunger at twelve days, and the test ended. The hatching rates and survival rates were calculated based on the results of eleven days; (c) Survival rates = survival number / hatching number x 100%.

Table 3: Abnormal Appearance and Abnormal Behaviour of Test Fish

Time (d)

Nominal concentration

Blank control

10% stock solution

20% stock solution

40% stock solution

80% stock solution

100% stock solution

0

30NB

30NB

30NB

30NB

30NB

30NB

1

30NB

29NB&1Dead

30NB

30NB

29NB&1Dead

29NB&1Dead

2

30NB

29NB&1Dead

30NB

30NB

29NB&1Dead

29NB&1Dead

3 (a)

29NB&1Dead

29NB&1Dead

30NB

30NB

28NB&2Dead

29NB&1Dead

4

29NB&1Dead

28NB&2Dead

29NB&1Dead

30NB

28NB&2Dead

29NB&1Dead

5

29NB&1Dead

28NB&2Dead

29NB&1Dead

29NB&1Dead

28NB&2Dead

29NB&1Dead

6

29NB&1Dead

28NB&2Dead

29NB&1Dead

29NB&1Dead

28NB&2Dead

29NB&1Dead

7

28NB&2Dead

28NB&2Dead

29NB&1Dead

29NB&1Dead

28NB&2Dead

29NB&1Dead

8

28NB&2Dead

28NB&2Dead

29NB&1Dead

29NB&1Dead

28NB&2Dead

29NB&1Dead

9

28NB&2Dead

28NB&2Dead

29NB&1Dead

29NB&1Dead

28NB&2Dead

29NB&1Dead

10

28NB&2Dead

28NB&2Dead

29NB&1Dead

28NB&2Dead

28NB&2Dead

29NB&1Dead

11

28NB&2Dead

28NB&2Dead

29NB&1Dead

28NB&2Dead

28NB&2Dead

29NB&1Dead

12 (b)

26NB&4Dead

27NB&3Dead

27NB&3Dead

25NB&5Dead

27NB&3Dead

26NB&4Dead

Note: NB: normal behavior and appearance; (a) Initiation of hatching on the third day; (b) The larvae in the control group began to die from hunger at twelve days, and the test ended.

Table 2: Hatching rate, survival rate and normal rate of the embryos and larvae at the end of the test (11 d)

Nominal concentration

Mean Measured concentration (µg/L)

Adjusted hatching rate, survival rate and normal rate (%) *

Hatching rate

Survival rate

Normal rate

Blank control

ND

100

100

100

10% stock solution

18.5

100

104

100

20% stock solution

34.6

103

96.7

100

40% stock solution

78.0

96.5

104

100

80% stock solution

145

100

104

100

100% stock solution

196

96.5

104

100

EC50 (µg/L, based on mean measured concentration)

> 196

> 196

> 196

95% CI (µg/L, based on mean measured concentration)

-

-

-

NOEC

100% stock solution (mean measured concentration 196 µg/L)

LOEC

≥ 100% stock solution (mean measured concentration 196 µg/L)

* Adjusted by Abbotts, formula. Adjusted hatching rate, survival rate and normal rate were calculated based on the results of eleven days.

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
EC50 (hatching, survival, malformation): >100% stock solution prepared at 100 mg/L after 24h stirring and filtration (mean measured concentration 196 ug/L)
LOEC (hatching, survival, malformations, length): > 100% stock solution at 100 mg/L after 24h stirring and filtration (mean measured concentration 196 ug/L)
NOEC (hatching, survival, malformations, length): 100% stock solution at 100 mg/L after 24h stirring and filtration (mean measured concentration 196 ug/L)
Executive summary:

In a 12 d semi-static toxicity test on embryo and sac-fry stages according to OECD Guideline 212 and GLP principles, fertilized eggs of Gobiocypris rarus (rare minnow) were exposed to the test substance at 10%, 20%, 40%, 80% and 100% of stock solution (prepared at 100 mg/L after 24h stirring and filtration) and an untreated control, in triplicate. Mean measured concentrations were 18.5, 34.6, 78.0, 145 and 196 ug/L, respectively. Hatching, survival, abnormal appearance and behaviour were monitored during the test and at the end of the test the total length of the fish was assessed. Hatching rate was 93.3 - 100%, overall survival was 93.0 - 100%. No abnormal appearance or behaviour was observed. No significant difference in total length was observed. The EC50 for hatching, survival and malformations was > 100% stock solution (mean measured concentration 196 ug/L); NOEC for hatching, survival, malformations and length was 100% stock solution (mean measured concentration 196 ug/L). The study met all validity criteria and is considered reliable without restriction.

Description of key information

In a 12 d semi-static toxicity test on embryo and sac-fry stages according to OECD Guideline 212 and GLP principles, fertilized eggs of Gobiocypris rarus (rare minnow) were exposed to the test substance at 10%, 20%, 40%, 80% and 100% of stock solution (prepared at 100 mg/L after 24h stirring and filtration) and an untreated control, in triplicate. Mean measured concentrations were 18.5, 34.6, 78.0, 145 and 196 ug/L, respectively. Hatching, survival, abnormal appearance and behaviour were monitored during the test and at the end of the test the total length of the fish was assessed. Hatching rate was 93.3 - 100%, overall survival was 93.0 - 100%. No abnormal appearance or behaviour was observed. No significant difference in total length was observed. The EC50 for hatching, survival and malformations was > 100% stock solution (mean measured concentration 196 ug/L); NOEC for hatching, survival, malformations and length was 100% stock solution (mean measured concentration 196 ug/L). The study met all validity criteria and is considered reliable without restriction.

Key value for chemical safety assessment

Additional information

NOEC: 196 ug/L, which is considered the maximum solubility of the test substance in test medium.

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