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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-02-11 to 2008-05-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples of the test concentrations and control were taken on days -1, 0 and at least weekly thereafter until end of exposure. Additionally, stock solutions were sampled and analysed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions were prepared with sterilised demineralised water. Ninety mL of stock solutions were used for 4 d. Stock solutions were stored at 6 +/- 2°C in the dark.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: Forellenzucht Worbis, Nordhäuserstr. 57, D-37339 Worbis

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
Unfertilized eggs and milt of Oncorhynchus mykiss (Rainbow trout) obtained from the breeder (see ahead) were used. After receipt the unfertilized eggs and milt were held at the temperature of 10°C for 10 minutes.

- Fertilization process:
The milt was added to the eggs. Gentle mixing was carried out for about 20 s. After 30 min without mixing water was added (1-2 cm layer) and gentle mixing was carried out for 1 min. After 5 min without mixing the eggs were rinsed with dilution water until clearness of the water. Thereafter the eggs were hardened under gentle flow-through (3.3 L/h) of dilution water for 1.5 h.

- Subsequent handling of eggs:
Eggs were exposed to the test solutions about 1.5 hours after the eggs had been fertilised. 35 eggs per incubation cup, 4 additional replicates each with 35 eggs in separate egg incubation cups surged around with dilution water alone were used for the determination of fertilization rate. At post-hatch day 6 larvae were random thinned to 15 individuals per replicate. At this day the individuals were released from the egg cup to the aquaria.

POST-HATCH FEEDING
- Start date: post-hatch day 15 (study day 52)
- Type/source of feed: Fresh cultures of Artemia salina (24 h old brine shrimp nauplii)
- Amount given: ad libitum
- Frequency of feeding: Food was added three to ten times daily
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
100 d
Remarks on exposure duration:
63 days post-hatch
Hardness:
Control: 59.3 +/- 4.27mg CaCO3 /L
5 µg a.i./L: 60.7 +/- 4.61 mg CaCO3 /L
135 µg a.i./L: 61.7 +/- 3.64 mg CaCO3 /L
405 µg a.i./L: 63.7 +/- 3.95 mg CaCO3 /L
1215 µg a.i./L: 73.9 +/- 0.00 mg CaCO3 /L
Test temperature:
10 +/- 2 °C for eggs, 12 +/- 2 °C for larvae
pH:
mean pH in the test media during the total test period for control and test item groups: 7.48 to 7.66; range: 7.21 to 8.03
Dissolved oxygen:
mean values during the total test period for control and test item groups: 99 to 100 % air saturation; range: 95 to 100 %
Nominal and measured concentrations:
nominal:
5 – 15 – 45 – 135 – 405 - 1215 µg active substance/L (corresponding to 17.2 – 51.5 – 155 – 464 – 1392 - 4175 µg test item/L)
mean measured concentrations
3.93 - 12.1 - 43.7 - 135 - 387 - 1199 µg active substance/L
The mean recovery rates for all concentrations were in the range of 81 – 100 %.
Details on test conditions:
TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): 8 cm diameter stainless steel pipes with plates perforated with holes (diameter 2 mm) on the bottom. These incubation cups were suspended in each replicate test aquarium. To facilitate circulation of water and keep eggs clean incubation cups were oscillated vertically (about 2 times per minute) with a lifting movement of approximately 4 cm in the test chamber by means of low rpm electric motor.
- Test vessel: glass aquaria
- Type: covered with perforated sheets with the beginning of swim-up
- Material, size, headspace, fill volume: glass aquaria, 12.5 cm x 14 cm x 21.5 cm with a volume of about 3.5 L
- Aeration: no
- Type of flow-through: Precision syringe pumps
- Renewal rate of test solution (flow rate): about 24 changes of test water per day
- No. of fertilized eggs/embryos per vessel: 35
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated drinking water was used for testing
- Total organic carbon: 0.605 mg/L (SD: 0.399 mg/L)
- Chlorine: < 0.01 mg/l
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Darkness for larvae until 1 week after hatch (except when being inspected), thereafter subdued light (0.1 - 10 µmol photons x m-2 x s-1 ) and 12 h photoperiod during this phase.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Egg/ embryo mortality, egg hatching, mortality of larvae and juvenile fish, abnormalties (e.g. hyperventilation, uncoordinated swimming, swim-up behaviour, atypical quiescence, and atypical feeding behaviour), measurements of fish size, wet/ dry body weight

VEHICLE CONTROL PERFORMED: no vehicle was used

RANGE-FINDING STUDY
- Test concentrations: 1.46, 14.6, 146 µg a.i./l
- Results used to determine the conditions for the definitive study: yes

POST-HATCH DETAILS
- Begin of post-hatch period: day 37 (91% of all fertilized and living embryos in the control groups have hatched)
- No. of hatched eggs (alevins)/treatment released to the test chamber: 15

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 4 x 35 eggs
- Removal of eggs to check the embryonic development on day no. 11
Reference substance (positive control):
not required
Duration:
37 d
Dose descriptor:
NOEC
Effect conc.:
0.135 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: egg hatch
Duration:
37 d
Dose descriptor:
LOEC
Effect conc.:
0.405 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: egg hatch
Duration:
100 d
Dose descriptor:
NOEC
Effect conc.:
0.135 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: length, dry weight, morphological and behavioural effects, post hatch success, overall survival
Duration:
100 d
Dose descriptor:
LOEC
Effect conc.:
0.405 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: length, dry weight, morphological and behavioural effects, post hatch success, overall survival
Details on results:
Biological parameters:
- Egg Fertilization Rate: The fertilization success ranged from 94 to 100 percent with the mean of 98 %
- 100 % Mortality of eggs was observed at 405 µg a.i./L at day 39. No egg survived at the highest test concentration
- Natural Egg Hatchability Rate: Corrected by the fertilization rate a natural egg hatchability rate of 95 % in the mean was reached. So, the minimum guideline requirement of > 66 % was reached.
- Egg Hatch: began on study day 35 in the control and all test item concentrations and continued until study day 39. Study day 37 was determined to be post hatch day 0 (PHD 0) with a control hatching rate of 91 %
- Swim-up : observed for a 15-day period on study days 53 to 67. Newly hatched fry began to swim up on study day 53 (PHD 16). On study day 67 (PHD 30) all larvae had swum up
- Post Hatch Success: The fry survival (post hatch success) on study day 42 (PHD 5) was 100 % in the control group, 100 % in the 5 µg a.i./L group, 98.5 % in the 15 a.i. µg/L group, 96.25 % in the 45 µg a.i./L group and 98.5 % in the 105 µg a.i./L group.
- Overall survival: 100 % in the control group, 98 % in the 5 µg a.i./L group, 100 % in the 15 µg a.i./L group, 97 % in the 45 µg a.i./L group and 95 % in the 105 µg a.i./L group. No statistically significant difference were found for this parameter
- Fry growth (length and dry weight): measured on study day 100 (PHD 63). Statistical analysis of data showed no significant differences between the controls and the test item concentrations for the dry weight
- Biomass loading: 28.5 mg per litre and day
- No biologically significant morphological and behavioural effects were observed in any tested replicate

Analytical monitoring:
The mean recovery rates for all concentrations were in the range of 81 – 100 %. Therefore all effect values are based on nominal concentrations.
Reported statistics and error estimates:
- One way analysis of variance (ANOVA) and Dunnett’s test were used for NOEC/LOEC calculations. When running a one way analysis of variance a normality test and an equal variance test were done first.
- Hatching data of days 37 - 39 were analysed with ANOVA and Dunnett’s test (SigmaStat)
- Dry weight was analysed with ANOVA (SigmaStat) after being transformed (Y = log Y, GraphPad Prism4)
- Post hatch survival (Post hatch success) was not statistically analysed because the data did not pass the normality test. The occurrence of 100 % values in several groups prevented the determination of normality.
- Mortality data of day 100 were analysed with ANOVA (SigmaStat)
Validity criteria fulfilled:
yes
Conclusions:
C8-18 AAPB caused significant effects on chronic toxicity (rainbow trout early life stage test, 62 days post hatch) at the nominal dosage levels 405 and 1215 µg/L. The overall NOEC (0-100 d) was 135 µg C8-18 AAPB/L based on the toxic endpoints egg hatch, time to hatch, time to swim-up, fry growth (expressed as length and weight), post hatch survival and overall survival.
Executive summary:

In a study conducted according to OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) and EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test), the 100– day chronic toxicity of C8 -18 AAPB to early life stage of rainbow trout (Oncorhynchus mykiss) was investigated under flow through conditions and analytical monitoring of test material concentrations. The mean recovery rates for all concentrations were in the range of 81 – 100 %. Therefore all effect values are based on nominal concentrations.

Fertilized eggs (4 x 35 eggs) were exposed to control and nominal concentrations of 0.005, 0.015, 0.045, 0.135, 0.405 and 1.215 mg active substance/L. The test system was maintained at 10 +/- 2 ºC (for eggs) and 12 +/- 2 °C (for larvae) and a pH of 7.48 to 7.66. The 37 – day NOEC and LOEC values, based on egg hatch, were 0.135 and 0.405 mg a.i./L respectively. The 100– day overall NOEC and LOEC values were 0.135 and 0.405 mg a.i./L, respectively. The effects included were length, dry weight, morphological and behavioural effects, post hatch success and overall survival.


This toxicity study is classified as reliable without restriction and satisfies the requirements of the guideline..

Results Synopsis

Test Organism Size/Age: freshly fertilized eggs
Test Type: Flow-through

100 d-LOEC: 0.405 mg a.i./

100 d-NOEC: 0.135 mg a.i./l
Endpoint(s) Effected: egg hatch, length, dry weight, morphological and behavioural effects, post hatch success and overall survival

Description of key information

One study for long-term toxicity to fish is available, investigating the 100– day chronic toxicity of C8 -18 AAPB conducted according to OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.135 mg/L

Additional information

An early life stage test as well as aprolonged 28-d toxicity study conducted with C8-18 AAPB are available.

 

In a study conducted according to OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) and EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test), the 100 day chronic toxicity of C8 -18 AAPB to early life stage of rainbow trout (Oncorhynchus mykiss) was investigated under flow through conditions and analytical monitoring of test material concentrations. The mean recovery rates for all concentrations were in the range of 81 – 100%. Therefore all effect values are based on nominal concentrations. The 37 day NOEC and LOEC values, based on egg hatch, were 0.135 and 0.405 mg a.i./L respectively. The 100 day overall NOEC and LOEC values were 0.135 and 0.405 mg a.i./L, respectively. The effects included were length, dry weight, morphological and behavioral effects, post hatch success and overall survival.

 

Additional data are available from a prolonged 28-d toxicity study conducted according to OECD Guideline 215 Draft 'Juvenile growth test: 28 d' (1992): Rainbow trouts (Oncorhynchus mykiss) were exposed to C8-18 AAPB under flow through conditions. A 28-d NOEC = 0.16 mg a.i./L nominal was determined based on growth (body length and mean body wet weight). At higher test concentrations of 0.5 and 1.6 mg a.i./L nominal, the growth rates could not be determined, since all fish died during the test period.

 

Conclusion

The obtained NOECs of 0.135 and 0.16 mg a.i./L of C8-18 AAPB were in the same range. The obtained results were considered to be valid for the AAPBs.

The NOEC of 0.135 mg a.i./L obtained from the long-term study including early life-stage was selected for further assessment and derivation of the PNEC.