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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from March 9th, 1989 to March 10th, 1989.
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to the OECD Guidelines n° 202.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:

Test material

Details on test material:
The substance examined was AF-366, a white solid. The test substance was received on September 27, 1988 from CIVO-TNO in a 500 ml polyethylene bottle with a red screw-cap, labelled ‘AF-366’. The solubility of the test substance in water was very low.
1 g of AF-366 was accurately weighed out and dissolved in 10 ml of tertiary butyl alcohol (TBA). In this way a concentrated solution of AF-366 in TBA
containing 100,000 mg.l-1 was prepared. From this solution 1.0 ml was diluted to 10 ml with TBA. In this way a concentrated solution of AF-366 in TBA containing 10,000 mg.1 was prepared. Test ‘solutions’ containing 1.0 and 10 mg of AF-366 per 1 of dilution water were prepared as follows: 10 µ1 of each concentrated solution indicated above were mixed with 100 ml of dilution water. An additional test ‘solution’ containing 100 mg.l-1 was prepared by mixing 100 µ1 of the concentrated solution in TBA containing 100,000 mg.l-1 with 100 ml of dilution water. It should be noted that the use of 100 µl of organic solvent per 100 ml of dilution water exceeds the allowable amount according the OECD Guideline no. 202 (the maximum allowable amount of an organic solvent is 100 mg per 1).
Some undissolved particles were seen after dosing in the test beakers containing the test ‘solutions’ of 10 and 100 mg.l-1.
In addition ‘best possible’ saturated solutions were prepared to avoid the use of an organic solvent. These solutions were prepared as follows: 1 g of
AF-366 was accurately weighed out in duplicate and each portion was added to 1 litre of dilution water. These suspensions were vigorously stirred with a magnetic stirrer for 24h at 20°C.
After this period one suspension was filtered through paper (S&S 595½), and portions of 100 ml of the filtrate were placed in each of four 150 ml bea
Portions of 100 ml of the other (unfiltered) suspension were placed in each of four 150 ml beakers. Care was taken to shake the suspension just before each addition to maintain the ratio between dissolved test substance and undissolved particles in the test beakers. This suspension was cloudy.
The composition and properties of AF-366 were not specified.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The organism used was the fresh-water crustacean Daphnia magna, cultured in
the laboratory under standard conditions, as described in NPR 6503 (ref. 2).
At the beginning of the test, the animals were less than 24 h old.

Study design

Test type:
Water media type:
Total exposure duration:
24 h

Test conditions

The hardness, expressed as CaCO3, was 212 mg.l-1.
Test temperature:
20 ± 1°C
between 7.8 and 8.2
Dissolved oxygen:
The oxygen concentrations of the test and control solutions were greater than 7.2 mg.l-1.
Nominal and measured concentrations:
The nominal composition is as described below:
Na+ 1.19 mmol.l-1
K+ 0.20
Ca2+ 1.36
Mg2+ 0.73
Cl- 2.72
S042- 0.73
HCO3- 1.39
This medium is prepared by addition of several saits to groundwater from a locality near Linschoten.
The groundwater contains several other trace elements (« 1 mg.l-1 ).
Media prepared from it have proved to be suitable for growing several species of water organisms.
The equilibrium pH of the medium, after aeration, should be 8.3 - 8.5, but usually is slightly less, namely 8.0 - 8.2.
The hardness, expressed as CaCO3, is about 210 mg.l-1.
The medium is prepared in large amounts (10,000 l) and its composition is checked by chemical analysis.
The actual composition of the batch used for this test was:
Na+ 1.35 mmol.l-1
K+ 0.27
Ca2+ 1.36
Mg2+ 0.76
Cl- 2.05
S042- 0.85
HC03- 1.62
The hardness, expressed as CaCO3, was 212 mg.l-1.
Details on test conditions:
The test was conducted in accordance with the OECD Guideline no. 202 (ref.
1); the exposure period was 24 h.
It was carried out in the period 9-10 March 1989.
Appropriate concentrations for the toxicity tests were determined in a preliminary range-finding test.
Five ‘solutions’ of AF-366 prepared as described in section 2.1, were tested; the ‘solutions’ used are given in Annex A, Table Al and A2. DSWL was
used as a blank control. The test was performed in 150 ml all-glass beakers,
each containing 100 ml of test solution. Four beakers containing 5 Daphnia
were used for each test or control solution.
The test lasted 24 h. The test and control solutions were not renewed. The
Daphnia were not fed. At t = 0 h and t = 24 h the pH and the oxygen concentration of the test and control solutions were measured. The test solutions were kept at 20 ± 1°C, under a 16 h light-8 h dark regime, and not aerated.
The immobile animals were counted after 24 h. At the same time the condition
of the mobile animals was compared with that of the control animals in the
The actual concentrations of test substance in the test solutions were not
determined by chemical analysis.

Results and discussion

Effect concentrations
0 h
Dose descriptor:
Effect conc.:
>= 0.119 mg/L

Applicant's summary and conclusion

The acute toxicity of AF-366 to the fresh-water crustacean Daphnia magna was
determined as laid down in the OECD Guideline no. 202, with an exposure period of 24 h.
The solubility of AF-366 in water was very low.
Test ‘solutions’, most of which were most probably saturated, were prepared
in several ways (see section 2.1).
Some undissolved particles were seen after dosing in the test beakers containing the test ‘solutions’ of 10 and 100 mg.l-1.
A suspension of 1000 mg.l-1 was stirred for 24h to prepare a ‘best possible’
saturated aqueous phase.
Except for two immobile animals in the concentration of 100 mg.l-1 and two
in the unfiltered ‘saturated solution’, none of the daphnids showed any visual sign of harm during the 24 h of exposure. The immobile animals had
suffocated in the test substance. It was therefore conciuded that AF-366 is not
acutely toxic to Daphnia magna at concentrations within its aqueous solubility.
No calculations on EC5O-values or the slope of the concentration-effect
curve could be made, because all the remaining animals were mobile after the
exposure period.
The concentrations quoted in this report refer to the product as supplied by
the sponsor.