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Short-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to relevant guidelines with clear and plausible results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes
Remarks:
The OECD Principles of Good Laboratory Practice

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
not applicable

Test solutions

Vehicle:
no
Details on test solutions:
The test substance was insoluble in water based on information supplied by the sponsor. Analytical confirmation of the actually dissolved concentration in test medium was not possible as no analytical method was available or could be developed with the necessary sensitivity to measure the soluble fraction of the test substance.
This was based on previous work on a similar compound, which showed that the water solubility was less than 10 µg/L (NOTOX Project 289979). The soluble fraction present in saturated solutions prepared during that project were not distinct from the background responses. All possible efforts were taken to improve the detection limit of the analytical method. However, these were not successful.
Preparation of a test solution at maximum saturation of the test substance in test medium started with a nominal concentration of 100 mg/L. This solution was treated with ultrasonic waves and subsequently magnetically stirred for 4 days. The resulting dispersion was then pre-filtered through a paper filter (Schleicher and Schuell 604) to remove the !arger undissolved test substance particles (ca. > 5µm), followed by filtration through a 0.45 µm membrane filter. The final test solution was a clear and colourless solution.
Note that the use of centrifugation to obtain the water-soluble fraction proved not to be successful as a consequence of the behaviour of the test substance in water, i.e. large amounts remained floating on the water surface.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Validity of batch: Frequent inspection of the cultures with respect to number of young, appearance of young and parental daphnia and global feeding behaviour.
- Characteristics: For the test selection of young daphnia with an age of < 24 hours.
- Start of each batch: With new-born animals, i.e. less than 3 days old, by placing about 250 of them into 10 litres of medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of medium: 18-22°C, constant within ± 1 °C
- Feeding: Daily, a suspension of fresh water algae.
- Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).




Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
250 mg/L expressed as CaCO3
Test temperature:
Blank control during the study: 19.6 - 20.4°C
pH:
At t=0h and t=48h
Control: 7.8 - 7.8
Test samples: 7.8 - 7.9
Dissolved oxygen:
At t=0h and t=48h [mg/L]
Control: 7.8 - 8.0
Test samples: 8.2 - 8.5
Salinity:
not applicable
Nominal and measured concentrations:
As the submission substance was insoluble in water (< 10µg/L), analytical verification was impossible due to sensitivity limitations. Therefore, in a limit test the nominal substance concentration of 100 mg/L in test medium was applied after filtration through a 0.45 µm membrane filter.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all glass
- static
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
Composition of medium M7:
ISO-medium: the following chemicals (analytical grade) are dissolved in freshly prepared ultra-pure water (tap water purified by reverse osmosis (milli-RO); Millipore Corp., Bedford, Mass., USA) (mg/L):
CaCl2.2H20 293.8
MgSO4.7H20 123.3
NaHCO3 64.8
KCI 5.8
Medium M7: trace elements, macro nutrients and vitamins are added to freshly prepared ISO-medium to reach the following concentrations:
Trace elements (mg/L):
B 0.125
Fe 0.05
Mn 0.025
Li, Rb and Sr 0.0125
Mo 0.0063
Br 0.0025
Cu 0.0016
Zn 0.0063
Co and I 0.0025
Se 0.0010
V 0.0003
Na2EDTA.2H20 2.5
Macro nutrients (mg/L):
Na2SiO3.9H20 10.0
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Vitamins (µg/L):
Thiamine 75.0
B12 1.0
Biotin 0.75
The hardness: 250 mg/L expressed as CaCO3 and the pH: 8.0 ± 0.2 after aeration.

OTHER TEST CONDITIONS
- Photoperiod: 16h day
- Aeration: No aeration of the test solutions
- Feeding: no
- Introduction of daphnids: directly after preparation of the test solutions



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilization, 24 and 48 hours

Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
EC50
Remarks:
Immobilization
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Immobility
The immobility data of the limit test are presented in Table 1. No daphnids became immobilised during the test period in the maximum soluble concentration in test medium.
In the blank-control not more than 10% of the daphnids became immobilised, see table 1.
Under the conditions of the present test, the test item induced no effects in Daphnia magna exposed to a filtered solution prepared at a loading of 100 mg/L (NOEC).
In conclusion: Owing to the extremely low solubility of the test item in water, concentration levels toxic for crustaceans could not be reached. Therefore, the 48h-EC50 for Daphnia magna exceeded the maximum solubility of the test item in water.



Results with reference substance (positive control):
48-hour Acute Toxicity Study in Daphnia magna with K2Cr2O7(NOTOX Project 341696).
The study procedures described in this report were based on the ISO International Standard 6341, the EEC directive 92/69, Part C.2. "Acute toxicity for Daphnia" and the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 4, 1984.
The reference test was carried out to check the sensitivity of the test system as used by NOTOX.
Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L and to a blank control. Ten daphnia were exposed per concentration.
The reference substance, potassium dichromate (K2Cr2O7, ad. 4864.0500, batch no. K28974764) was obtained from Merck, Darmstadt, Germany.
For details see table 2.
The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at NOTOX.
The 24h-EC50 was 1.3 mg/L with 95% fiducial limits of 1.0 to 1.8 mg/L.
The 48h-EC50 was 0.55 mg/L with 95% fiducial limits of 0.48 to 0.69 mg/L.
The test described above was performed under GLP.

Any other information on results incl. tables

Table 1: Acute immobilisation of daphnia after 24 and 48 hours in the limit test.

Concentration

Test substance

Nominal (mg/L)

Vessel

Number

Number

Daphnia

exposed

Response at 24 h

Response at 48 h

number

%

number

%

Blank-control

A

10

1

10

1

10

B

10

0

0

0

0

100*

A

10

0

0

0

0

B

10

0

0

0

0

* 0.45 µm filtrate.

Table 2: reference substance

Acute immobilization of daphnia alter 24 and 48 hours in the reference test with potassiumdichromate:

Concentration
K2Cr2O7 (mg/L)

Number Exposed

% immobile

24h

% immobile

84h

Expected response (%)After 48 hours1

Minimal                                Maximal

0.00

10

0

0

0

102

0.10

10

0

0

0

10

0.18

10

0

0

0

10

0.32

10

0

0

0

30

0.56

10

0

50

0

100

1.0

10

0

100

40

100

1.8

10

100

100

100

100

1Based on historical data of the previous years (n>60).

2A maximum response of 10% does not invalidate the results of the test.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present test, the test substance induced no effects in Daphnia magna exposed to a filtered solution prepared at a loading of 100 mg/I (NOEC)
Executive summary:

The effects of the submission substance on Daphnia magna straus had been tested according to the ISO International Standard 6341: "Water quality Determination of the inhibition of the mobility of Daphnia magna Straus - Acute toxicity test, Third edition, 1996-04-01 and equivalent to OECD 202 and EU-method C.2. The test fulfilled all the validity criteria and is of reliability category 1.

Because of the insolubility of the submission substance in water (< 10 µg/L), a nominal concentration of 100 mg/L had been used after filtration (0.45 µm membrane filter) and analytical monitoring had not been possible becaus of sensitivity limitations. At the test concentration, no immobilization of daphnids could be observed. The nominal test concentration of 100 mg/L of the submission substance is therefore the NOEC (48h).