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Ecotoxicological information

Long-term toxicity to fish

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fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Oct - 02 Dec 1998
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
adopted 1992
according to guideline
EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)
Version / remarks:
adopted 1986
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: Samples of each replicate at each treatment level were collected at test initiation (before the introduction of test embryos), at test termination and once per week during the duration of the test.
- All samples were collected from the approximate midpoint of the test solutions.
- Sample storage conditions before analysis: Samples were analysed immediately after sampling.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)
- Method: For each test concentration, a stock solution was prepared at 7 or 8 day intervals by dissolution of the test substance in DMF.
- Concentration of vehicle in test medium (stock solution and final test solution): 0.05 mL/L
Test organisms (species):
Pimephales promelas
Details on test organisms:
- Common name: Fathead minnow
- Source: Fertilised embryos were obtained from Bio International, 36204 Nantes, France. The source of these embryos was Aquatic Research Organisms, Hampton NH, USA
- Age of embryos at test initiation: < 24 h

- Start date: when hatch for any test level was at least 90% or 48 h after first hatch
- Type/source of feed: live brine shrimp nauplii (Artemia salina)
- Frequency of feeding: 3 times per day (week days), twice daily (weekends). From approx. 10 days post hatch: live brine shrimp (≤ 48 h) ad libitum.
Test type:
Water media type:
Limit test:
Total exposure duration:
35 d
15 - 48 mg/L CaCO3
Test temperature:
23.8 - 25.5 °C
6.5 - 7.5
Dissolved oxygen:
5.6 - 7.6 mg/L
> 60% air saturation value
Nominal and measured concentrations:
Nominal: 0, 0.01, 0.03, 0.09, 0.27 and 0.81 mg/L
Mean measured: < LOQ, 0.01, 0.03, 0.10, 0.29 and 0.84 mg/L
Details on test conditions:
- Embryo cups: The embryos were incubated in egg incubation cups constructed from glass cylinder of 44 mm internal diameter. A nylon mesh (pore size 450 µm) support was attached using silicone sealant to the lower end. The egg cups were gently oscillated in the test solutions by means of a rocker arm apparatus driven by a 2 rpm electric motor.
- Fry enclosures: During approx. 10 days post hatch, the fry were contained in an enclosure (inside the relevant aquarium) made of a nylon screen attached to a glass petri dish.
- Test vessel: rectangular aquaria
- Material, size, fill volume: glass, 20 L, 15 L
- Aeration: none; the dilution water was saturated by air bubbling in the head tank.
- Type of flow-through (e.g. peristaltic or proportional diluter): For each test solution (each replicate), a 25 mL plastic syringe in conjunction with a Harvard syringe pump was calibrated to deliver approximately 2.5 µL/min of the relevant stock solution into a chemical mixing chamber which also received approximately 50 mL/min of dilution water via a peristaltic pump. The homogenised test solution then flowed into the relevant test aquarium.
- Renewal rate of test solution (frequency/flow rate): approx. 5 aquarium volumes/day
- No. of fertilized eggs/embryos per vessel: 80
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

- Source/preparation of dilution water: tap water filtered through active charcoal and diluted with deionised water
- Total organic carbon: < 1 mg/L
- Particulate matter: < 1 mg/L
- Metals: Ca 12.1, Mg 3.6, K 0.2, Na 16.9, other metals < 0.005 to 0.6
- Pesticides: < 0.01 to 10 µg/L
- Chloride: 5.8 mg/L
- Alkalinity: 0.4 - 0.8 mmol/L
- Ca/Mg ratio: 3.3:1
- Conductivity: 126.5 - 143.5 µS/cm
- Intervals of water quality measurement: Temperature, pH and dissolved oxygen were recorded in each test aquarium at test initiation and termination and three times a week during the test period. The water temperature in one test aquarium was continuously recorded throughout the test period. Total hardness, alkalinity and specific conductance were measured from one replicate of the highest and lowest test substance concentrations, the solvent control and the dilution water control, at test initiation and termination and, at least, once a week during the test period.

- Photoperiod: 16 h light, 8 h dark with 30 min transition
- Light intensity: 410 - 741 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Hatching time (from test start until hatching began the embryos in each cup were examined daily and mortalities recorded)
- Percentage of hatching
- Survival of fry from hatch to test end
- Wet weights and total lengths of surviving fry at test termination


No details given.

- Begin of post-hatch period: Hatching was first observed on day 3 of the test and was considered complete on day 4 of the test when at least 90% of all remaining embryos had hatched in each egg cup of each test group.
- No. of hatched eggs (alevins)/treatment released to the test chamber: 40
- Release of alevins from incubation cups to test chamber: 10 days post hatch
Reference substance (positive control):
35 d
Dose descriptor:
Effect conc.:
0.01 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
35 d
Dose descriptor:
Effect conc.:
0.03 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
35 d
Dose descriptor:
other: MATC
Effect conc.:
0.017 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: geometric mean of the range 0.01 - 0.03 mg/L
Details on results:
- Average percent survival in the exposed groups at test end: 98.8, 100, 96.3 and 91.3% at the mean measured concentrations of 0.01. 0.03, 0.10 and 0.27 mg/L, respectively. 100% mortality was recorded at 0.85 mg/L before test termination.
- Mortality in the controls: None
- Abnormal observations: At test termination, no other sub lethal toxic effects were observed except at the second highest concentration of 0.27 mg/L where a low number of physically malformed fish were observed in each replicate aquarium (1 in replicate A and 2 in replicate B).
- Incidents in the course of the test which might have influenced the results:
(1) The water pumps and syringe injectors feeding each test aquarium stopped during one night (29 Nov 1998) and the test solutions remained under static conditions for approx. 11 h before being restarted the following morning. This was not thought to have any influence on the test results as the test solution volumes were maintained in each aquarium, the test substance is known to remain stable in dilution water for periods greater than 11 h, each pump stopped and was started at the same time and no signs of stress were observed among the test fish at the end of this period.
(2) On two occasions during the test period (28 Oct 1998 and 12 Nov 1998) technical problems with the diluting system resulted in a decrease in water hardness below the range of 40-48 mg/L recommended by the EPA guideline. The lowest value observed was 15 mg/1. On both occasions corrective measures were taken and additional measurements were made the following day which showed that the water hardness was increasing to normal levels. This deviation was not thought to have influenced the test results in any significant way as all the biological criteria were fulfilled in the control groups and all of the test aquaria were exposed to the same variations.
- Measured test substance concentrations at test start: 100 - 111% of nominal values; exception: replicate B at nominal 0.27 mg/L with only 11% of nominal, but no difference in biological effects compared to replicate A was observed.
- Measured test substance concentrations once per week and at test end: 83 - 115%

Percentage embryo hatch: Compared to the control, the mean percentage of hatchability was significantly reduced at the three highest concentrations (α = 0.01) of 0.10, 0.27 and 0.85 mg/L with 94.4, 90.7 and 0% hatch, respectively. There was no significant difference between the control and the two lowest test concentrations of 0.01 and 0.03 mg/L with 97.6 and 98.2% hatch, respectively.

Survival of fry to test termination: No significant difference in percentage survival of the fathead minnow fry to test termination was observed between the solvent control group and the two test concentrations of 0.01 mg/L (98.8% survival) and 0.03 mg/L (100% survival). The three highest concentrations were excluded from the statistical analysis of this parameter.

Wet weights and total lengths: Both the fry mean wet weight and mean total fry length of the 0.03 mg/L group were significantly reduced compared to the solvent control (α = 0.01). No significant difference between the solvent control and either wet weight or total length was observed at the lowest test concentration of 0.01 mg/L.

Table 1: Validity criteria for OECD 210.

Criterion from the guideline


Validity criterion fulfilled

The dissolved oxygen concentration should be >60% of the air saturation value throughout the test.

 5.6 - 7.6 mg/L


The water temperature should not differ by more than ± 1.5 °C between test chambers or between successive days at any time during the test, and should be within the temperature ranges specified for the test species (Annex 2).

Differences observed between test
chambers and between successive days of the test were always less than 1.5°C


The analytical measure of the test concentrations is compulsory.

 Analytical measurements were performed


Overall survival of fertilised eggs and post-hatch success in the controls and, where relevant, in the solvent controls should be greater than or equal to 70% and 75%, respectively.

 Control hatching success: 98.2%


control hatching success: 99.4.2%

Post-hatch success in both controls: 100%


Validity criteria fulfilled:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

NOEC (35 d) = 0.01 mg/L (mean measured, Pimephales promelas, OECD 210)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
Effect concentration:
0.01 mg/L

Additional information

The long-term effects of the substance towards fish were examined in a 35-day early-life stage toxicity test with fathead minnow (Pimephales promelas) under flow-through conditions. The study was carried out according to GLP and OECD 210. Based on mean measured concentrations of the test substance (100 – 111% of nominal values at test start and 83 – 115% at test end), a NOEC (35d) of 0.01 mg/L and a LOEC of 0.03 mg/L were determined.