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Long-term toxicity to fish

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long-term toxicity to fish, other
Prolonged acute fish toxicity test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Feb 2000 to 2 Mar 2000
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
other: OECD Guideline 203
Study was extended to 21 days
GLP compliance:
not specified
Not stated, but appears to be conducted similar to GLP studies
Analytical monitoring:
Details on sampling:
- Sample storage conditions before analysis: samples were analyzed immediately and processed within 2 hrs, analytical standards were kept in acetonitrile at 0 - 4°C.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 40 µL of test compound was dissolved in 22 mL of acetone. After that, 356 mL of MilliQ water was added. The solution was pumped into a premix flask. Fresh stock solution was prepared daily.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): < 100 mg/L
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Common name: zebra fish
- Source: Agrojardin, Avda. Cantabria 4, San Sebastian de los Reyes, 28050 Madrid, Spain
- Length at study initiation: 2 ± 1 cm

- Food type: Nutrafin
- Amount: 2% mean bodyweight
- Frequency: once every three days

- Acclimation period: 14 days
- Acclimation conditions (same as test or not): water temperature, photoperiod and feed were similar to those in test
- Type and amount of food: Nutrafin, 10% of bodyweight
- Feeding frequency: once a day every working day
- Health during acclimation (any mortality observed): no mortality observed
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
49.52 - 60.95 mg/L as CaCO3
Test temperature:
20 ± 1°C
6.33 - 7.41
Dissolved oxygen:
44 - 85.8% of saturation
Nominal and measured concentrations:
Measured: 57.78 ± 31.85 ng/mL (equivalent to 57.78 µg/L)
Details on test conditions:
- Test vessel: Aquaria
- Material, size, fill volume: Glass, 40 cm x 25 cm x 20 cm, 15 L
- Aeration: Dilution water tank was aerated with oil-free air; exposure tanks were not aerated
- Type of flow-through (e.g. peristaltic or proportional diluter): peristaltic
- Flow rate: 1.32 L/hr
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 10

- Source/preparation of dilution water: Madrid tap water, dechlorinated using sodium thiosulphate, purified with charcoal
- Nitrites: 0.003-0.070 mg NO2-
- Conductivity: 142.6 - 213 µS/cm
- Intervals of water quality measurement: every 3 - 4 days (hardness, pH and dissolved oxygen were determined twice weekly and temperature was determinded daily)

- Photoperiod: 12 hrs light/12 hrs dark

EFFECT PARAMETERS MEASURED: Mortality, behaviour and toxic symptoms

- Test concentrations: Acetone was used as a vehicle to obtain a testing concentration above the water solubility (37.2 ± 11.7 ng/mL) of LAB. Note that all concentrations in the study were reported in units of ng/mL, which is equivalent to µg/L. The standard unit for aquatic toxicity data is µg/L and so this unit was used to report the results in this summary.
Reference substance (positive control):
Key result
21 d
Dose descriptor:
Effect conc.:
>= 57.78 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: behaviour, toxic symptoms, mortality
Remarks on result:
other: no toxic effects observed
21 d
Dose descriptor:
other: LC0
Remarks on result:
other: above water solubility limit
Details on results:
No mortality was observed in the test substance aquarium. One incidental non-treatment related mortality was observed in the control group. Another fish was removed from the control group due to attack by other fish. No toxic symptoms were observed in any of the test aquaria. Note that individual homologues were also tested. Phenyl C10, C12 and C18 also resulted in no toxic symptoms for the duration of the study.
Validity criteria fulfilled:
Based on the findings, the 21-day NOEC zebra fish exposed to LAB was determinded to be ≥ 57.78 µg/L, and the LC0 was above the water solubility limit.
Executive summary:

The effects of LAB on zebra fish (Danio rerio) was studied in a flow-through toxicity test. The study was conducted according to OECD TG 203. It was not compliance with GLP but appears to be conducted similar to GLP studies. Two groups of ten zebra fish were exposed in an extended acute test to a test solution of LAB for 21 days. This high exposure was obtained by using acetone as a vehicle. The measured test concentration was 57.78 µg/L, which was above the water solubility of 37.2 µg/L (determined in a water solubility test conducted concurrently). No mortality, behavioural changes, or toxic symptoms were noted in the exposure groups.

Based on the findings, the 21 -day NOEC for zebra fish was determined to be ≥ 57.78 µg/L, the LC0 was above the water solubility limit of LAB.

Description of key information

A 21-day long-term toxicity test of the test subtsance to freshwater fish (Danio rerio) was conducted according to OECD guideline No.203. The NOEC based on behaviour, toxic symptoms and mortality was determinded to be > 57.78 µg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
57.78 µg/L

Additional information

An OECD 203 study with zebra fish (Danio rerio) was conducted in which the exposure period was extended to 21 days. Fish were exposed to LAB in a flow-through system at a limit concentration of 58 µg/L using acetone as a solvent. Fish were fed with Nutrafin once every three days during the test. No mortality was observed in the treatment solutions during the study. No toxic symptoms were observed. The 21-day NOEC is therefore greater than the limit dose of 58 µg/L.

The lack of fish toxicity is consistent with expectations. Overall, LAB has very low water solubility and in a 14-day study with fish there were no effects at the limits of water solubility. In fact, in short-term tests conducted by Gledhill et al. (1991), fish were unaffected even at nominal concentrations of 1000 mg/L and with LAB visibly floating on the surface of the test vessels. Furthermore, short-term studies conducted with aquatic invertebrates and algae indicated no doseresponsive effects at the water solubility limit. Finally, a 21-day long-term study in Daphnia was conducted and resulted in a LOAEC of 0.015 mg/L (15 µg/L). Based on the generally greater sensitivity of Daphnia than fish, the lack of bioaccumulation potential, the available fish, invertebrate and algae data, and in accordance with Annex IX, Section 9, column 2 of the REACH regulation, a long-term toxicity study in fish is not deemed necessary.

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