[No public or meaningful name is available]

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March from the 12th to the 14th, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Remarks:

validated analytical method

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

updated 13th April 2004

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

April 1996

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentrations, samples were taken from each of the four vessel of the treatment and the control groups at the start and at the end of the test.

Vehicle:

no

Details on test solutions:

The test solution was prepared by mechanical dispersion. An amount of 0.03 g test item was dissolved in 300 ml test medium (OECD medium) using approx. 15 min ultrasonic dispersion resulting in a concentration of 100 mg/l. The test solution was freshly prepared in the test laboratory just before introduction of the test animals (start of the experiment).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection), Gödöllő, Hungary.
- Breeding: the Daphnia are bred in the testing laboratory.
- Age: less than 24 h old at the beginning of the test.
- Feeding during test: test animals were not fed during the exposure.
- Food type: concentrated algal suspension of Pseudokirchneriella subcapitata during the holding.
- Animal health: apparently healthy animals were used in this test with a known history.

ACCLIMATION
- Acclimation period: brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.
- Acclimation conditions: test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

41.5 mg/l (as CaCO3); due to the chemical properties of the used test medium, hardness resulted out of the recommended range of 140 - 250 mg/l, however there was no effect observed in behaviour of daphnids and therefore this value was considered to be acceptable and does not affect the results of the test.

Test temperature:

19.5 - 19.9 °

pH:

7.42 - 7.97

Dissolved oxygen:

7.36 - 8.48 mg/l

Nominal and measured concentrations:

100 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker; volume app. 50 ml.
- No. of organisms per vessel: 5 animals per vessel.
- No. of vessels per concentration: four replicates.
- No. of vessels per control: the test medium without test item added was used as control solution; four replicates.
- Biomass loading rate: at least 4 ml test solution/animal.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: based on the results of preliminary solubility tests, the test item could not be completely dissolved at a concentration level of 100 mg/l in ISO medium, Elendt M4 medium or natural water (recommended test media according to OECD 202), therefore OECD medium (reconstituted water, according to OECD 201) was used as dilution water in the experiment (in which the test item can be diluted completely at 100 mg/l).
- Stock solution 1 (macro nutrients): NH4Cl 15.0 mg/, MgCl2 (anhydrous) 5.6 mg/l (this corresponds to the concentration of 12.0 mg/l of MgCl2 × 6 H2O (cf. OECD no. 201 guideline)), CaCl2 × 2 H2O 18.0 mg/l, MgSO4 × 7 H2O 15.0 mg/l, KH2PO4 1.6 mg/l.
- Stock solution 2 (iron): FeCl3 × 6 H2O 64.0 μg/l, Na2EDTA × 2 H2O 100.0 μg/l.
- Stock solution 3 (trace elements): H3BO3 185.0 μg/l, MnCl2 × 4 H2O 415.0 μg/l, ZnCl2 3.0 μg/l, CoCl2 × 6 H2O 1.5 μg/l, CuCl2 × 2 H2O 0.01 μg/l, Na2MoO4 × 2 H2O 7.0 μg/l.
- Stock solution 4 (bicarbonate): NaHCO3 50.0 mg/l.

OTHER TEST CONDITIONS
- Photoperiod: the light-dark cycle during the test was 16 hours light (artificial illumination) and 8 hours darkness.

EFFECT PARAMETERS MEASURED
Daphnia were observed for immobility or mortality by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.

RANGE-FINDING STUDY
- Test type: non-GLP preliminary static test.
- Test concentrations: 100 mg/l (limit concentration).
- N. of organisms: ten daphnids (divided into two replicates).
- Duration: 48 hours.
- Control: a control group run concurrently.
- Results used to determine the conditions for the definitive study: no immobility or any sub-lethal effect was detected in the treated or in the control groups during the 48-h exposure period.

REFERENCE SUBSTANCE
For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study with reference item Potassium dichromate was: 05 – 06 March 2014.

VALIDITY CRITERIA
- The immobilisation rate of the introduced Daphnia should not exceed 10 % in the control group.
- The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/l in control and test vessels.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

There was no immobilisation observed in twenty daphnids exposed to each test item treated and control group. Further no abnormal behaviour or appearance of test animals was noted.
The 48-h NOEC was determined to be 100 mg/l.

TEST CONCENTRATIONS
The test item was not detected in the untreated control group. In the treated group measured concentrations of test item were 98 % of the nominal at the start and 99 % at the end of the test. The measured test item concentration remained within ± 20 % of the nominal during the test period of 48 hours, therefore biological results are given as nominal concentration.

Results with reference substance (positive control):

EC50 (24h): 1.31 mg/l (95 % confidence limits: 1.15 - 1.60 mg/l).

Immobilization of the test animals

Test Group	Replicate	Number of treated animals	Number of immobilised animals
			24 h	48h
Control	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
100 mg/l	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0

Measured concentrations of test item in OECD Medium

Test Group	Mean of the measured concentrations (mg/l) with the 95 % confidence interval (n=4)		Measured concentration in % of the nominal
	0 h	48 h	0 h	48 h
Control	not detected	not detected	-	-
100 mg/l	98.20 (RSD: 2 %)	99.25 (RSD: 1 %)	98	99

pH-values measured during the experiment

Test Group	Replicate	pH
		0 h	48h
Control	1	7.75	7.86
	2		7.90
	3		7.93
	4		7.97
100 mg/l	1	7.42	7.87
	2		7.89
	3		7.81
	4		7.88

Dissolved Oxygen Concentrations measured during the experiment

Test Group	Replicate	Dissolved Oxygen Concentration [mg/l]
		0 h	48h
Control	1	8.48	8.07
	2		8.13
	3		7.91
	4		7.94
100 mg/l	1	7.9	7.91
	2		7.93
	3		7.36
	4		7.84

Temperatures measured during the experiment

Test Group	Replicate	Temperature [°C] 0h
		0 h	48h
Control	1	19.5	19.9
	2		19.9
	3		19.7
	4		19.8
100 mg/l	1	19.5	19.7
	2		19.8
	3		19.8
	4		19.8

Validity criteria fulfilled:

yes

Remarks:

in the control not more that 10 % of the daphnids were immobilised and the dissolved oxygen was higher than 3 mg/l in control and test vessels

Conclusions:

EC50 (48h) > 100 mg/l (nominal)

Executive summary:

The substance was assessed using Daphnia magna in a 48-hour Acute Immobilisation Test. Based on results obtained in a preliminary experiment, organisms were exposed to a single concentration of 100 mg/l (limit concentration), over a period of 48 hours. A concurrent control group was run. A static test was performed as the test item was previously shown to be stable in the test medium for 48 hours. The analytically measured test item concentration remained within ± 20 % of the nominal during the test period of 48 hours; therefore, biological results are based on the nominal concentration.

There was no immobilisation observed in the twenty daphnids exposed to either the test item or in the control group. In addition to immobility, no abnormal behaviour or appearance of test animals was noted. The 48-h NOEC was determined to be 100 mg/l.

Conclusion

EC50 (48h) > 100 mg/l (nominal)

Description of key information

Not harmful/toxic for short-term exposure to invertebrates.

EC50 (48h) > 100 mg/l (nominal)

Additional information

The substance was tested for short-term toxicity to invertebrates on Daphnia magna. The test was conducted according to the OECD guideline 202. Based on results obtained in a preliminary experiment, organisms were exposed to a single concentration of 100 mg/l (limit concentration).No immobilisation was observed in the twenty daphnids exposed to either the test item or in the control group. In addition to immobility, no abnormal behaviour or appearance of test animals was noted. The analytically measured test item concentration remained within ± 20 % of the nominal during the test period of 48 hours; therefore, biological results are based on the nominal concentration.