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Ecotoxicological information

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:
From March 23rd to April 28th, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD guidance document no. 23, GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF DIFFICULT SUBSTANCES AND MIXTURES, adopted Dec. 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
The content of the test item in the test vessels was measured at the beginning and the end of the study and once a week during the course of the test. Six measurements were performed.

Sample preparation concentration 10 mg/L
After adding 6 g of NaCl, 50 mL test solution was extracted two times with the solvent ethyl acetate (15 and 10 mL), the organic phase was collected into a 20-50 mL pear shaped flask, after drying with Na2SO4 and evaporated to dryness. The residue was then reconsti-tuted using 5 mL of ethyl acetate.
An aliquot (2 mL) of the sample was added to 10 mL volumetric flask, 1 mL of 0.1 % phenolphthalein in methanol solution and 1 mL of 0.1 M phenyltrimethylammonium hydroxide was added and the flask was filled up to 10 mL with ethyl acetate. Internal standard solution can be added to the test solution before filling the flask. Twofold enrichment was achieved during the sample preparation.

Sample preparation concentration 4.6 mg/L and Control
After adding 6 g of NaCl, 100 mL test solution was extracted two times with the solvent ethyl acetate (15 and 10 mL), the organic phase was collected into a 20-50 mL pear shaped flask, after drying with Na2SO4 and evaporated to dryness. The residue was then reconstituted using 5 mL of ethyl acetate.
An aliquot (2 mL) of the sample was added to 10 mL volumetric flask, 1 mL of 0.1 % phenolphthalein in methanol solution and 1 mL of 0.1 M phenyltrimethylammonium hydroxide was added and the flask is filled up to 10 mL with ethyl acetate. Internal standard solution can be added to the test solution before filling the flask. Fourfold enrichment was be achieved during the sample preparation.

Vehicle:
no
Details on test solutions:
The water-accommodated fraction (WAF) was prepared for the test. This was done by mixing the nominal load 100 mg/L (100.025 mg/L - 102.000 mg/L) with the corresponding amount of dilution water and stirring vigorously for 24 ±1 hours. The concentrations to be used in the test were prepared by dilution of this WAF. Therefore, samples of the WAF were taken carefully after a settling time of approx. 15 minutes to avoid any carry-over of undiluted test item.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Fertilised eggs of Danio rerio HAMILTON-BUCHANAN were used in the test.
The original animals were obtained from Umweltbundesamt (UBA) and used for breeding and production of eggs.

Danio rerio is routinely used for breeding and toxicity tests. The fish are kept following SOP 115 005 04 in the current edition.
Vessels glass aquaria
Medium chlorine-free tap water with a copper concentration below 0.05 mg/L (composition see Annex 2: Analysis of Drinking Water)
Food three times a working day with warm-water fish food
Cleaning and medium renewal at least once per week
Photo period 12/12 hours, using neon tubes
Temperature 23 ±1 °C

For production of eggs, one day before the start of the test, six aquaria with 10 L were prepared and equipped with spawn dishes and artificial plants. In each aquarium, two adult males and one adult female were set. On the next morning, the eggs were carefully removed. After microscopical observation, the eggs to be used in the test were separated.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
35 d
Hardness:
106.8 mg CaCO3/L
Test temperature:
25.0 – 25.9 °C
pH:
7.3 - 8.0
Dissolved oxygen:
6.1 - 8.0 mg/L
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Nominal: 4.6 and 10 mg/L
The geometric mean of the measured concentrations was 92 % (4.25 mg/L) of the nominal concentration for treatment 4.6 mg/L and 96% (9.65 mg/L) of the nominal concentration for treatment 10 mg/L. Therefore the biological results were based on the nominal concentrations.
Details on test conditions:
Duration: 35 days
Treatments tested: 4.6 and 10 mg/L
Concentration stock solutions 100 mg/L nominal concentration, stock WAF
46 mg/L dilution of stock WAF (dilution factor 2.2)
Flow stock solutions 1 mL/min
Flow dilution water 3.7 mL/min day 0 – day 14
4 mL/min day 14 – day 35
Test vessels: stainless steel aquaria (volume 13 L)
Replicates four replicates, each containing 13 L test solution and 30 eggs
Control four replicates, each containing 13 L dilution water and 30 eggs
Temperature: 25.0 – 25.9 °C
Photo period: 12/12 hours using neon tubes
Aeration: from day 14 on
Feeding: starting with the hatching on day 5 with powdered flake food until day 34
Reference substance (positive control):
no
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
number hatched
Details on results:
Results on the control (0 mg/L - 4 replicates)
Mean hatching rate : 86%
Mean post-hatch survival: 85%
Mean fish weight: 3.29 mg
Mean fish lenght: 9.45 cm

Results at 4.6 mg/L (4 replicates)
Mean hatching rate : 89%
Mean post-hatch survival: 81%
Mean fish weight reduction compared to control: 1.7%
Mean fish lenght reduction comapred to control: 4.0%


Results at 10 mg/L (4 replicates)
Mean hatching rate : 83%
Mean post-hatch survival: 50%
Mean fish weight reduction compared to control: 37.4%
Mean fish lenght reduction comapred to control: 37.0%

None of the surviving fish showed abnormal behaviour.
Results with reference substance (positive control):
Not performed
Validity criteria fulfilled:
yes
Conclusions:
The substance showed adverse effects on survival, weight and lenght of fish after hatching at a concentration of 10 mg/L, which was considered to be the LOEC.
The NOEC for long-term exposure to fish was determined to be 4.6 mg/L.
No observations were made which might cause doubts concerning the validity of the study outcome. All validity criteria were met.
The result of the test is considered valid.
Executive summary:

The toxicity of the test item in the Early-Life Stage Toxicity Test against Danio rerio was tested under flow-through conditions. One valid experiment was performed using the nominal concentrations 4.6 mg/L and 10 mg/L test item. For each treatment, 30 fish eggs were exposed to the test item. 30 fish eggs were used in the control. 4 replicates were performed for each concentration.

The concentration 10 mg/L showed significant toxicity whereas at the concentration 4.6 mg/L no toxicity could be observed.

 

The content of the test item in one replicate per concentration was measured once a week using GC/MSD. The geometric mean of the measured concentrations was 92 % of the nominal concentration for the treatment 4.6 mg/L and 96 % of the nominal concentration for the treatment 10 mg/L.

Therefore, the determination of the results was based on the nominal concentrations.


The following results for the test item DIACID 1550were determined:

 

Endpoint

NOEC

LOEC

Hatching

≥ 10 mg/L

> 10 mg/L

Mortality

4.6 mg/L

10 mg/L

Weight

4.6 mg/L

10 mg/L

Length

4.6 mg/L

10 mg/L

Description of key information

Based on read-across to the free acid, that was already tested in a fish early life stage study, according to OECD 210, the NOEC (35d-Danio Rerio) for the potassium salt is set to 5.1 mg/L for survival, weight and length.

Key value for chemical safety assessment

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

Additional information