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 fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 09-27, 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 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 7346-1 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 1: Static Method)

Deviations:

no

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 with surviving fish and the control according to the schedule below.
Frequency at t=0 h, t=24 h and t=96 h
Volume 2 ml
Storage Samples were stored in a freezer until analysis.

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 10 mg/l (292 mg was weighed into 25 litres 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 test concentration in test medium. The final test solutions were all clear and increasingly orange with increasing concentration.

Test organisms

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: carp
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands
- Length at study initiation: 2.5 +/- 0.2 cm
- Weight at study initiation: 0.47 +/- 0.08 g
- Method of breeding: single parent-pair breeding
- Feeding during test: no feeding from 24 hours prior to the test and during the total test period

ACCLIMATION
- Acclimation period: at least 12 days after delivery
- Acclimation conditions: same as test concerning test water. Water qualitiy parameters were kept within the optimum for the fish species. No data on illumination and areation.
- Type and amount of food: pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands). No data on amount.
- Feeding frequency: daily
- Health during acclimation (any mortality observed): In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

180 mg CaCO3/l

Test temperature:

21.2-22.1 °C

pH:

7.5-8.0

Dissolved oxygen:

5.4-9.5 mg/l

Nominal and measured concentrations:

nominal concentrations: 0 (controls), 1.0, 1.8, 3.2, 5.6 and10 mg/l
mesured concentrations: Analysis was performed on samples taken from the test groups that were considered essential for determination of the toxicity values (nominal 10 and 5.6 mg/l). Analysis at the start of the test showed measured concentrations of 108 % and 107 % at the nominal concentrations of 5.6 and 10 mg/l, respectively. During the exposure period the measured concentration remained constant (104-107% of nominal).

Details on test conditions:

TEST SYSTEM
- Test vessel:10 litres, all-glass, containing 9.8 litres of test solution
- Aeration: none
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.34 g/l


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: no
- Photoperiod: 16 hours photoperiod daily
- Light intensity: low light intensity


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
mortality and other effects; at 2, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 and every morning from day 1 to observe for any dead or severely distressed fish. Dead fish were removed when observed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

- Range finding study
- Test concentrations: 0, 0.09, 0.86, 8.6, 86 mg/l
- Results used to determine the conditions for the definitive study: see Table 1.

Reference substance (positive control):

yes

Remarks:

pentachlorophenol (PCP), 0, 0.10, 0.22, 0.46 mg/l

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

water free

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL 5.6-10 mg/L

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

water free

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: none
- Other biological observations: 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 96 h: 0.10 mg/l: 0%; 0.22 mg/l: 60%; 0.46 mg/l: 100%
- 96h-LC50: 0.20 mg/l (95 % confidence interval between 0.16 and 0.32 mg/l)

Reported statistics and error estimates:

The LC50 could not be determined using the maximum likelihood estimation method with the probits of the percentages of dead fish as function of the logarithms of the corresponding concentrations (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition). This was because there was no concentration between the highest concentration (A) at which 0% mortality and the lowest concentration (B) at which 100% mortality occurred. Instead, the LC50 was calculated as (AB)½, with A and B being limits of the 95% con-fidence interval.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Criteria of OECD Guideline 203: Mortality of control animals <10%; concentration of dissolved oxygen in all test vessels >60% saturation; concentration of test substance ≥80% of initial concentration during test.

Conclusions:

For classification purposes: 1 mg/l < 96h-LC50 ≤ 10 mg/l