Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[3-(dimethylamino)propyl]amino]sulfonyl derivs., acetates

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:

September from 11th to 13th, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the start of the intervals samples from vessels without daphnids and at the end of the intervals samples from vessels with daphnids were analysed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test concentrations were prepared by diluting a stock solution of the test substance in M4 medium. A fresh stock solution was made on each day of test solution renewal.
The test substance (200.4 and 200.9 mg) was weighed in and made up with 2 litres M4 medium. The mixture was treated with an ultrasonic bath for about 2 minutes and afterwards stirred on a magnetic stir plate for about 10 minutes at 20 ± 2 °C. The stock solution was diluted with M4 medium to make each test solution concentration. After preparation, all nominal concentrations were clear and blue. Foam was observed on the surface of all freshly prepared solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna STRAUS
- Source: the clone of Daphnia magna STRAUS 1820 used in the study was originally obtained from the Institut National de Recherche Chimique Appliquée, France, in 1978. From this date on this clone was cultured and bred continuously in the testing laboratory.
- Age at study initiation: 2 - 24 h (at least 3rd brood progeny).
- Age of parent animals: 2 - 4 weeks.
- Feeding during test: none.

CULTURE CONDITIONS
- Photoperiod: 16 hours light, 8 hours dark
- Water quality: same as test water.
- Temperature: 20 ± 2 °C
- Diet: unicellular algae Desmodesmus subspicatus, cultured continuously at the test facility
- Feeding: daily
- Renewal of the culture water: twice weekly.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

2.20 - 3.20 mmol/l

Test temperature:

20.3 - 21.2 °C

pH:

7.9 - 8.1

Dissolved oxygen:

8.6 - 8.9 mg/l

Nominal and measured concentrations:

1.0, 2.2, 5.0, 10, 22, 50 and 100 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: flat-bottom glass test tubes (nominal volume: 20 ml)
- Test volume: 10 ml
- Aeration: no
- Loading: 0.5 animals/ml.
- Renewal rate of test solution: water renewal after 24 hours because the test substance precipitates in the presence of impurities in water.
- No. of organisms per vessel: 5 daphnids.
- No. of test animals per concentration: 20
- No. of vessels per concentration: 4 replicates.
- No. of vessels per control: 4 replicates.
- Additional replicates: 1 additional replicate without animals per test concentration and control for measurement of the pH and oxygen content at the beginning of each interval. This replicate was discarded after measurement.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic fresh water (Elendt M4). The composition of this M4 medium is given in OECD guideline 202.
- Total hardness: 2.20 – 3.20 mmol/l
- Acid capacity up to pH 4.3: 0.80 – 1.00 mmol/l
- Molar ratio Ca:Mg: about 4 : 1
- pH value: 7.5 – 8.5
- Conductivity: 550 - 650 μS/cm
- Aeration: prior to use the M4 medium was aerated for approximately 24 hours until saturation with oxygen was reached.

OTHER TEST CONDITIONS
- Photoperiod: 16 h day: 8 h night.
- Illumination: artificial light, type warm white (OSRAM L58 W31).
- Light intensity: 20 - 850 Lux at a wavelength of 400 - 750 nm.

EFFECT PARAMETERS MEASURED
Immobilisation after 0, 24 and 48 h.

TEST CONCENTRATIONS
The test concentrations were selected on the basis of a preliminary range finding test (experimental conduct in accordance with GLP but without a GLP Status) conducted as part of the study. EC50 range derived from 48 hour range finding test: 10 - 100 mg/l.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 101 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

EFFECTS
Two immobilized daphnids were observed in the 100 mg/l concentration at 48 hrs. Please note that this minor level of effect is within the accepted tolerance for control performance.

ANALYTICAL CONCENTRATION
A slight adsorption was measured also in the control group. This is considered to be caused by components released to the test water by the daphnids rather than from any contamination by the test substance in the control group. Therefore a background correction was carried out for the mean values. A background correction for the individual determinations was considered to be not appropriate, since the variability between the measurements was relatively high. In the three highest test concentrations all analytically determined concentrations of the test substance in the freshly prepared test solutions and in the 24 hour old test solutions were in the range of 90 – 106 % of the nominal concentration, even if the background correction was considered.In the concentrations < 10 mg/l the analytically determined concentrations were markedly below the nominal concentration and the concentrations and/or decreased markedly over the 24 hour period. However, since the lower concentrations were not relevant for the determination of the EC-values, the low measured values had no influence on the results of the study.

Results with reference substance (positive control):

The EC50(24h) of the control substance potassium dichromate was 1.26 mg/l.
The result is within the range of 0.6 - 2.1 mg/l (ISO 6341) and indicates that the culture of D. magna used in this study is responding normally to toxic stress.

Number of mobile Daphnids

Number of mobile Dahnids

Nominal conc. (mg/l)	Replicate 1			Replicate 2			Replicate 3			Replicate 4			Σ
Time, h	0	24	48	0	24	48	0	24	48	0	24	48	24	48
0 (control)	5	5	5	5	5	5	5	5	5	5	5	5	20	20
1	5	5	5	5	5	5	5	5	5	5	5	5	20	20
22	5	5	5	5	5	5	5	5	5	5	5	5	20	20
5	5	5	5	5	5	5	5	5	5	5	5	5	20	20
10	5	5	5	5	5	5	5	5	5	5	5	5	20	20
22	5	5	5	5	5	5	5	5	5	5	5	5	20	20
50	5	5	5	5	5	5	5	5	5	5	5	5	20	20
100	5	5	4	5	5	5	5	5	5	5	5	4	20	18

ANALYTICAL CONCENTRATION

Nominal conc. (mg/l)	Mean over all measurements (mg/l)	Mean values after background correction: -0.16 mg/l
0	0.16	0
1.0	0.31	0.15
2.2	0.80	0.64
5.0	2.98	2.82
10	8.06	7.90
22	21.14	20.98
50	49.29	49.13
100	100.6	100.44

Nominal conc. (mg/l)		0	1	2.2	5	10	22	50	100
At start (0h), fresh test solution	330/331 nm	0.4	0.27	0.92	4.16	8.81	22.43	48.98	100.97
	602-/603 nm	0.18	0.23	0.91	3.57	8.65	22.21	48.58	101.47
	Mean	0.29	0.25	0.92	3.87	8.73	22.32	48.78	101.22
	% of nominal	-	25	42	77	87	101	98	101
At the end of interval, 24 h old test solution	330/331 nm	0.29	0.16	0.34	1.89	6.62	19.83	48.68	97.59
	602-/603 nm	0.2	0.17	0.37	1.98	6.72	19.88	47.09	95.44
	Mean	0.25	0.17	0.36	1.94	6.67	19.86	47.89	96.52
	% of nominal	-	17	16	39	67	90	96	97
At the start of interval (24 h), fresh test solution	330/331 nm	-0.12	0.64	1.16	3.87	9.32	22.24	52.86	103.49
	602-/603 nm	0.05	0.65	1.34	3.95	9.47	22.32	52.69	104.45
	Mean	-0.04	0.65	1.25	3.91	9.4	22.28	52.78	103.97
	% of nominal	-	65	57	78	94	101	106	104
At the end of interval (48h), 24 h old test solution	330/331 nm	0.17	0.16	0.71	2.21	7.42	20.21	47.16	99.94
	602-/603 nm	0.11	0.2	0.63	2.16	7.43	19.99	48.27	101.39
	Mean	0.14	0.18	0.67	2.19	7.43	20.1	47.72	100.67
	% of nominal	-	18	30	44	74	91	95	101

-: not determined

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

EC50 (48 h) > 101 mg/l (meas. arithm. mean)

Executive summary:

The potential toxicity of the test substance to Daphnia magna was examined according to OECD guideline 203 and Commission Directive 92/69/EEC. Nominal test concentrations tested were 1.0, 2.2, 5.0, 10, 22, 50 and 100 mg/l. The test was carried out in a semistatic system with a water renewal after 24 hours because the test substance precipitates in the presence of impurities in water, which was expected to cause a decrease of the analytically determined concentrations. The aqueous phase of the tested concentrations was clear and blue over the exposure period with foam on the water surface of all test solutions.

The analytically determined concentrations of the test substance in the test medium in the test concentrations > 22 mg/l were within a range of ± 10 % of the nominal value. In the test concentrations < 10 mg/l the analytically determined values were markedly lower than the nominal concentration most likely due to a possible precipitation of the test substance in the presence of impurities in water. However, since these concentrations were not relevant for the determination of the EC values, this had no influence on the results of the study.

Two immobilized daphnids were observed in the 100 mg/l concentration at 48 hrs; this minor level of effect is within the accepted tolerance for control performance.

Conclusion

EC50 (48 h) > 101 mg/l (meas. arithm. mean)

Description of key information

EC50 (48 h) > 101 mg/l (meas. arithm. mean)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

The potential toxicity of Basic Blue 140 was assayed in Daphnia magna, following the procedures outlined in the OECD guideline 203. Nominal test concentrations tested were 1.0, 2.2, 5.0, 10, 22, 50 and 100 mg/l. The test was carried out in a semistatic system with a water renewal after 24 hours because the test substance precipitates in the presence of impurities in water, which was expected to cause a decrease of the analytically determined concentrations. The aqueous phase of the tested concentrations was clear and blue over the exposure period with foam on the water surface of all test solutions.

The analytically determined concentrations of the test substance in the test medium in the test concentrations > 22 mg/l were within a range of ± 10 % of the nominal value. In the test concentrations < 10 mg/l the analytically determined values were markedly lower than the nominal concentration most likely due to a possible precipitation of the test substance in the presence of impurities in water. However, since these concentrations were not relevant for the determination of the EC values, this had no influence on the results of the study.

Two immobilized daphnids were observed in the 100 mg/l concentration at 48 hrs; this minor level of effect is within the accepted tolerance for control performance.