Reaction product of 3-[4-(2-aminoethylamino)-6-(4-sulfoanilino)-1,3,5-heterocycle-2-ylamino]benzensulfonic acid, ammonia water, sodium chloride and {mixture of [poly(1~4)chlorosulfonylphthalocyaninato-N29,N30,N31,N32]copper(II), [poly(1~3)chlorosulfonyl (tribenzo[b,g,l]pyrido[2,3-q]-5,10,15,20-tetraazaporphyrinato-N21,N22,N23,N24)]copper(II),[poly(1~2)chlorosulfonyl(dibenzol[b,g(or b,l)dipyrido[2,3(or 3,2)-l,q (or g,q)]-5,10,15,20-tetraazaporphyrinato-N21,N22,N23,N24)] copper(II) and [monochlorosulfonyl(benzo[b]tripyridol[2,3(or 3,2)-g,lq]-5,10,15,20- tetraazaporphyrinato- N21,N22,N23,N24)] copper(II)

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:

12 January 2009 to 19 February 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19 August 2008; Date of signature: 04 March 2009

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification : H-CB sodium salt
Description : blue solid
Lot number : MB-2
Date received : 04 December 2008
Storage conditions : room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Theoretical concentration of 8.0 to 11 mg/l, nominal concentration of 100 mg/l .
- Sampling method: The test material concentrations in the test samples were determined spectrophotometrically using an external standard.
- Sample storage conditions before analysis: Ambient temperature in light and dark conditions for approximately 48 hours.Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was dissolved directly in reconstituted water.
- Controls: Potassium dichromate

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: In-house laboratory cultures.
- Age at study initiation (mean and range, SD): <24 hours old
- Weight at study initiation (mean and range, SD): Not stated.
- Length at study initiation (length definition, mean, range and SD): Not stated.
- Feeding during test
- Food type: Algae (Chlorella sp.)
- Frequency: Daily.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

250 mg/l as CaC03

Test temperature:

Approximately 20°C, recorded daily using a Hanna Instruments HI 93510 digital thermometer.

pH:

8.0, recorded daily using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Dissolved oxygen:

8.3 - 9.2 mg O2/1, recorded daily using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Salinity:

Not applicable, fresh water.

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56 and 100 mg/I.
Measured concentratins: 87 - 96% of nominal.

Details on test conditions:

TEST SYSTEM
- Test vessel
- Type: Covered.
- Material, size, headspace, fill volume: 250 ml glass jars containing approximately 200ml of test preparation.
- Type of flow-through: Not applicable.
- Renewal rate of test solution: Not applicable.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
i) Stock Solutions
a) CaC12.2H20 11.76 g/l
b) MgS04.7H20 4.93 g/l
c) NaHC03 2.59 g/l
d) KCI 0.23 g/l

ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaC03.

- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
An estimate of the EC50 value at 24 hours was given by inspection of the immobilisation data.
The EC50 value at 48 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999). Probit analysis is used where two or more partial responses to exposure are shown.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study: Definitive test: 10, 18, 32, 56 and 100 mg/l.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: No observations made.
- Other biological observations:
- Mortality of control:The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd =0.21).
- Other adverse effects control: None
- Abnormal responses:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test preparations were observed to be blue solutions with increasing intensity with increasing concentration for the test media throughout the test. The control media was observed to be a clear, colourless solution throughout the test.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration. The slopes and their standard errors of the response curves at 24 and 48 hours were 8.1 (SE = 1.7) and 8.6 (SE = 1.8) respectively.The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd =0.21).
- Mortality: 100% immobilisation after 48 hours. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given inthe attachments section.
- EC50/LC50: EC50 > 3 hrs = > 3.2 mg/l; 24 hrs = 0.82 mg/l and 48 hours 0.71 mg/l.

Reported statistics and error estimates:

The ECso values and associated values at 48 hours the were calculated by the Maximum Likelihood-Probit calculation method using the ToxCalc computer software package. Probit analysis is used where two or more partial responses to exposure are shown.

Any other information on results incl. tables

Range-finding Test

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in Table 1. below:

Table 1.

Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 per Replicate)
	24 hours	48 hours
Control	0	0
0.10	0	0
1.0	0	0
10	0	0
100	1	1

No immobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/l. Ten percent immobilisation was observed at 100 mg/l which was considered to be due to natural causes.

Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/l.

Initial experiment

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the initial experiment are given in Table 2. below:

Table 2.

Cumulative Immobilisation Data in the Initial Experiment

Nominal Concentration (mg/l)		Cumulative ImmobilisedDaphnia (Initial Population: 5 per Replicate)
		24 hours			48 hours
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0
100	R1	1	1	0	2	7	35
	R2	0			1
	R3	0			4
	R4	0			0

R1 - R4 =Replicates 1 to 4

Definitive Test

Immobilisation data

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 3. below:

Table 3.

Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 5 per Replicate)
	24 hours				48 hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0
18	0	0	0	0	2	1	3	15
32	0	0	0	0	0	0	0	0
56	0	0	0	0	4	5	9	45
100	0	0	0	0	4	6	10	50

R1 - R2 =Replicates 1 and 2

The relationship between percentage immobilisation and concentration at 48 hours is given in Attachment 1 in the attachments section. Inspection of the immobilisation data at 24 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:

Time (h)	EC50	95% Confidence limits (mg/l)
24 48	>100 91	-* -*

* Data not suitable for calculation of 95% confidence limits

The No Observed Effect Concentrations after 24 and 48 hours exposure were 100 and 32 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration. Immobilisation of 15% was observed at 18 mg/l after 48 hours however, no immobilisation was observed at 32 mg/l. Given the low immobilisation at 18 mg/l, and the fact that 0% was observed at 32 mg/l, the No Observed Effect Concentration is considered to be 32 mg/l. The immobilisation at 18 mg/l was considered to be due to natural causes and/or handling stress. This was not considered to have affected the integrity of the study.

The slope and the standard error of the response curve at 48 hours was 2.2 (SE = 0.9).

Verification of test concentrations

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 87% to 96% of nominal value and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.

Positive Control

The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Attachment 1 in the attachments section

. Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results:

Time (h)	EC50	95% Confidence limits (mg/l)
3 24 48	>3.2 0.82 0.71	- 0.71 – 0.94 0.61 – 0.81

The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration. The slopes and their standard errors of the response curves at 24 and 48 hours were 8.1 (SE = 1.7) and 8.6 (SE = 1.8) respectively. The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mgll (sd =0. 21).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour ECso value of 91 mg/l. The No Observed Effect Concentration at 48 hours was 32 mg/l.

Executive summary:

Introduction. A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods. Following a preliminary range-finding test and an initial experiment, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 10, 18, 32, 56 and 100 mg/I for 48 hours at a temperature of approximately 20°C under static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 hours. A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/I for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results. The 48-Hour EC50 for the test material to Daphnia magna based on nominal test concentrations was 91 mg/1. The No Observed Effect Concentration was 32 mg/l.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 87% to 96% of nominal value and so the results are based on nominal test concentrations only. The 48-Hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.71 mg/l with 95% confidence limits of 0.61 - 0.81 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

Conclusion The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour ECso value of 91 mg/l. The No Observed Effect Concentration at 48 hours was 32 mg/l.