NO. 701 BASE

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 February 2011 - 02 March 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Description: Yellow-brown powder
Batch: 00120R

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter was retained for possible analysis of the residue.

Frequency: at t=0 h and t=48 h
Volume: 2 ml from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Additionally, reserve samples of 2 ml were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of the substance tested was a yellow-brown powder. The test substance was not completely soluble in test medium at the loading rate initially prepared.

Preparation of the test solutions started with prepared individually loading rates of 1.0, 10 and
100 mg/l applying a 2-day period of magnetic stirring to achieve maximum dissolution in test medium. The obtained aqueous mixture was filtered through a 0.45 µm membrane filter (Whatman, rc 55) and the clear and colourless filtrates were used as test concentrations. The lowest test concentration was prepared by subsequent dilution of the filtrate prepared at the loading rate of 1.0 mg/l in test medium.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

none

Test conditions

Hardness:

180 mg/l expressed as CaCO3

Test temperature:

The temperature of the test medium was 20.8 °C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 19.3 and 20.1°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

pH:

7.6-8.0

Dissolved oxygen:

9.4-9.8

Nominal and measured concentrations:

See table below in materials and methods. The measured concentration in the sample taken from the highest test concentration at the start of the test was 18 mg/l. During the exposure period the measured concentration remained constant (97% of initial). Based on these results the effect parameters were expressed in terms of the initially measured concentrations.

Details on test conditions:

- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 100 ml, normal headspace, 80 ml
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 for the highest concentration and 2 for the lower concentrations
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts:
CaCl2.2H2O : 211.5 mg/l
MgSO4.7H2O: 88.8 mg/l
NaHCO3 : 46.7 mg/l
KCl : 4.2 mg/l
The hardness: 180 mg/l expressed as CaCO3 and the pH: 7.7 ± 0.3 after aeration.
- Culture medium different from test medium: yes, for culturing M7 medium is used
OTHER TEST CONDITIONS

- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : EC50 t=24, 48 h and NOEC
TEST CONCENTRATIONS

- Spacing factor for test concentrations: 10
- Test concentrations:control and 0.45 µm filtered solutions prepared at loading rates of 1.0, 10 and 100 mg/l. In addition a ten-fold dilution of the filtrate prepared at a loading rate of 1.0 mg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

18 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: .

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 18 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: .

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 18 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: .

Details on results:

No immobility was observed at any concentration tested.

Results with reference substance (positive control):

The 24h-EC50 was 0.77 mg/l with a 95% confidence interval between 0.72 and 0.86 mg/l.
The 48h-EC50 was 0.57 mg/l with a 95% confidence interval between 0.52 and 0.66 mg/l.

Reported statistics and error estimates:

No EC50 could be calculated because the test substance proved to be non-toxic (EC50 > maximum soluble concentration tested).

Any other information on results incl. tables

Acute immobilisation of daphnids after 24 and 48 hours

Test substance Filtrate prep. at (mg/l)	Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
			number	Total %	number	Total %
control	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
10% of 1.0	A  B	5 5	0 0	0	0 0	0
1.0	A  B	5 5	0 0	0	0 0	0
10	A  B	5 5	0 0	0	0 0	0
100 (18)	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0

( ) between brackets: Initial concentration

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance did not induce acute immobilisation of Daphnia magna at 17.8 mg/l after 48 hours of exposure (NOEC).
The 48h-EC50 was beyond the range tested, i.e. exceeded an initially measured concentration of 18 mg/l.
Due to low solubility of the test substance the concentrations that might be toxic to Daphnia magna could not be reached.