1,1'-{(phenylmethanediyl)bis[(3-substituted benzene-4,1-diyl)diazene-2,1-diyl{1-[3-(dimethylamino)propyl]-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridine-5,3-diyl}]}dipyridinium chloride lactate salts

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:

February 09-25, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study compliant with TG guideline and conducted under GLP in recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test samples for analysis were taken from all test concentrations and the control according to the schedule below.
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.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preparation of test solutions started with a test solution of 100 mg/l (115.4 mg test substance was weighed in 1 litre of test medium; a correction factor of 1.16 was used to correct for water and minor impurities). The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and increasingly orange with increasing concentration.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain: Straus, 1820
- Source: In-house laboratory culture with a known history
- Age at study initiation (mean and range, SD): < 24 hours, from parental daphnids of more than two weeks old
- Method of breeding: About 250 newborn daphnids, i.e. less than 3 days old, placed into 5 litres of medium in an all-glass culture vessel. Maximum age of the cultures was 4 weeks. Renewal of the cultures after 7 days of cultivation (half of the medium twice a week)
- Feeding during test: no feeding

ACCLIMATION
- Acclimation period: not indicated

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg CaCO3/l

Test temperature:

20.5 - 21.7 °C

pH:

7.7-8.0

Dissolved oxygen:

8.8-9.0

Nominal and measured concentrations:

nominal concentrations: 0 (controls), 4.6, 10, 22, 46 and 100 mg/l
Analysis of the samples taken at the start of the test showed that measured concentrations were in agreement with nominal (83 – 105%). Concentrations measured after 48 hours all remained stable at more than 80% of initial. Nevertheless, average exposure concentrations (geometric mean) were calculated based on the fact that the recovery for 46 mg/l decreased below 80% relative to nominal (see Table 1).

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 80 ml of test solution
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic, adjusted ISO medium (as described in test guideline, however 1.4-fold diluted to reach a lower hardness) formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: the test was performed in the dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality); at 24 hours and at 48 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations: 0, 0.09, 0.86 and 8.6 mg/l
- Results used to determine the conditions for the definitive study: see Table 2

Reference substance (positive control):

yes

Remarks:

potassium dichromate, 0, 0.18, 0.32, 0.56, 1.0, 1.8 mg/l

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

water free

Basis for effect:

mobility

Remarks on result:

other: 95% CL 9.2-14 mg/l

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

water free

Basis for effect:

mobility

Remarks on result:

other: 95% CL 43-129 mg/l

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

4.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

water free

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: none
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Mortality after 24 h: control: 0%, 0.18-0.32 mg/l: 0%, 0.56 mg/l: 50%, 1.0-1.8 mg/l: 100%
- Mortality after 48 h: control: 5%, 0.18 mg/l: 15%, 0.32 mg/l: 45%, 0.56-1.8 mg/l: 100%
- EC50 (24 h): 0.55 mg/l (95% confidence interval 0.50-0.63 mg/l)
- EC50 (48 h): 0.30 mg/l (95% confidence interval 0.25-0.35 mg/l)

Reported statistics and error estimates:

The EC50-value was calculated at 24 and 48 hours of exposure from the probits of the percentages of affected daphnids and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition). For parameters of the Probit analysis see Table 1

Any other information on results incl. tables

Table 1 Parameters of Probit analysis

Parameter	24 h	48 h
index of regression significance	g=0.30	g=0.12
chi-squared	5.48	13.96
degrees of freedom	10	18
regression line	log10(conc.)=1.65+(probit-4.69)/2.07	log10(conc.)=1.08+(probit-5.07)/3.72

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Criteria of OECD Guideline 202: In the control, no daphnids became immobilised. The oxygen concentration at the end of the test was ≥ 3 mg/l in control and test vessels.

Conclusions:

For classification purposes: 10 mg/l < 48h-EC50 ≤ 100 mg/l