Cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, sodium salts

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 - 28 May 1993.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD Guidelines for the Testing of Chemicals (1984) No . 201 "Alga, Growth Inhibition Test"

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

EEC Commission Directive 87/ 302/ EEC.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

An aliquot of each test sample was diluted in culture media to a final theoretical test material concentration of 10 mg/l.

Vehicle:

no

Details on test solutions:

Direct dispersion in culture medium.
200 mg of test material was dispersed in culture media and the volume adjusted to 1 litre to give a 200 mg/l stock solution. 500 ml of this stock solution was added to 500 ml of algal suspension to give the test concentration of 100 mg/l.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Name: Scenedesmus subspicatus (CCAP 276/ 20)
Source: Culture Centre of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Cumbria, U.K.
Pre-culture: Sterile culture medium was inoculated from a master culture of Scenedesmus subspicatus and incubated under continuous illumination (approximately 7000 lux) and aeration at 21 °C to give an algal suspension in log phase growth characterised by an absorbance of 0.702@ 665nm). The suspension was diluted to an absorbance of 0.027 prior to use. This suspension had a mean cell density of 3.69e+4 cells/ ml.

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period specified.

Test temperature:

24 °C

pH:

7.8 - 9.4

Dissolved oxygen:

Not specified

Salinity:

Not applicable-freshwater study

Conductivity:

Not specified

Nominal and measured concentrations:

Verification of test concentrations showed that the measured test concentrations were near nominal. Therefore it is considered justifiable to estimate the Ec50 values in terms of nominal test concentrations only.

Details on test conditions:

Exposure Conditions
Test Vessels: 250 ml conical flasks containing 100 ml test solution and loosely stoppered to reduce evaporation. All flasks were incubated and shaken (approximately 100 rpm) in an orbital shaker.

Experimental design: 1 test concentration, 6 replicates, plus 1 control, 3 replicates
This experimental design conforms to a "Limit Test" to confirm no effect on the growth of algal cells at the maximum test concentration of 100 mg/l.

Method of initiation: 500 ml algal pre-culture were added to 500 ml of sterile nutrient medium containing appropriate amounts of test material.

Photoperiod: Continuous (approximately 7000 lux).
Temperature: 24 °C.

Test concentration: 100 mg/l, six replicates. This test concentration was assigned following a preliminary range-finding study whereby the test organisms were exposed to the following test concentrations for a period of 96 hours: 0.0010, 0.010, 0.10, 1.0, 10 and 100 mg/l. The results showed no impairment of growth at 100 mg/l indicating the choice of test concentration employed.

Medium renewal: None.

Duration of exposure: 96 hours.

pH: The pH of each test and control culture was measured at 0 and 96 hours.

Measurement of growth: Samples were taken at 0, 24, 48, 72 and 96 hours and cell densities determined by direct counting of cells with the aid of a haemocytometer.
Spectrophotometer readings could not be used to monitor growth as the test material solutions gave an absorbance reading at the same wavelength used to monitor the growth of algal cells (665 nm).

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

From the data available, it is clear that neither the growth (r) or the biomass (b) of Scenedesmus subspicatus (CCAP 276/20) was affected by the presence of 100 mg/ 1 of the test material, JPR Blue 100, over the 96 hour exposure period.

Accordingly the following results were determined from the data:
EbC50 (72h): > 100 mg/l
EbC50 (96h): > 100 mg/l
ERC50 (0 - 24 h): > 100 mg/l

Where EbC50 is the test concentration that reduces biomass by 50% and ErC50 is the test concentration that reduced specific growth rate by 50%.

One way analysis of variance was performed on the area under the growth curve data for the control and 100 mg/l test group. There were no statistically significant differences (P ≥ 0.05) between the control and 100 mg/1 test group and therefore the ''No Observed Effect Concentration" (NOEC) is given as ≥ 100 mg/ l.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.

The following data:
Mean cell density of control @ 0h: 3.69e+4 cells/ ml
Mean cell density of control @ 72h: 7.32e+5 cells/ml
Mean cell density of control @ 96h: 1.04e+6 cells/ ml

show that the cell concentration of the control cultures increased by a factor of 20 during the test in line with the OECD Guideline that states that the enhancement must be at least by a factor of 16 after 72 hours.

Observations
All test and control cultures were inspected microscopically at 96 hours. There were no abnormalities detected in any of the control or test cultures at 100 mg/l.

Results with reference substance (positive control):

Not specified

Reported statistics and error estimates:

Not specified

Mean Cell Densities from the Exposure of Scenedesmus subspicatus to JPR Blue 100 for a Period of 96 hours

Concentration (mg/l)	pH	Mean Cell Densities* (cells/ml)					pH
	0 h	0 h	24 h	48 h	72 h	96 h	96 h
ControlR1            R2            R3	7.9 7.9 7.9	3.70 x 10^4	2.16 x 10^5	3.95 x 10^5	7.32 x 10^5	1.04 x 10^6	9.4 9.4 9.4
100      R1            R2            R3            R4            R5            R6	7.8 7.8 7.8 7.8 7.8 7.8	4.01 x 10^4	2.08 x 10^5	4.06 x 10^5	7.27 x 10^5	1.05 x 10^6	9.3 9.3 9.3 9.4 9.3 9.3

R₁– R₆= Replicate 1 – 6

*Cell densities represent the mean number of cells per ml calculated from the mean of cell counts from 3 fields of view for each of the replicate flasks.

 

Inhibition of Growth from the Exposure of Scenedesmus subspicatus to JPR Blue 100 for a Period of 72 and 96 hours

Concentration (mg/l)	Area under Curve @ 72h	% Inhibition	Area under Curve @ 96h	% Inhibition	Growth Rate (0-24 h)	% Inhibition
Control 100	2.12 x 10^7 2.11 x 10^7	- 1	4.16 x 10^7 4.14 x 10^7	- 0	0.073 0.069	- 6

 

Validity criteria fulfilled:

not specified

Conclusions:

These results are based on nominal test concentrations.
EbC50 (72h): > 100 mg/l
EbC50 (96h): > 100 mg/l
ErC50 (0-24h): > 100 mg/l
"No Observed Effect Concentration" (NOEC) ≥ 100 mg/l

Executive summary:

TEST MATERIAL: JPR Blue 100

 

TEST SPECIES: Scenedesmus subspicatus(CCAP 276/ 20)

 

TEST TYPE: Algal growth inhibition.

 

TEST CONDITIONS: Cultured under continuous illumination on an orbital shaker at 24 °C for 96 hours according to OECD Guideline No. 201 and EEC Commission Directive 87/302/EEC.

 

RESULTS

These results are based on nominal test concentrations.

EbC50 (72h):               > 100 mg/l

EbC50 (96h):               > 100 mg/l

ErC50 (0-24h):             > 100 mg/l

"No Observed Effect Concentration" (NOEC):≥100 mg/l

 

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.

Description of key information

These results are based on nominal test concentrations.

EbC50 (72h): > 100 mg/l

EbC50 (96h): > 100 mg/l

ErC50 (0-24h):           > 100 mg/l

"No Observed Effect Concentration" (NOEC):≥100 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

TEST MATERIAL: JPR Blue 100

 

TEST SPECIES: Scenedesmus subspicatus(CCAP 276/ 20)

TEST TYPE: Algal growth inhibition.

TEST CONDITIONS: Cultured under continuous illumination on an orbital shaker at 24 °C for 96 hours according to OECD Guideline No. 201 and EEC Commission Directive 87/302/EEC.

 

RESULTS

These results are based on nominal test concentrations.

EbC50 (72h): > 100 mg/l

EbC50 (96h): > 100 mg/l

ErC50 (0-24h):           > 100 mg/l

"No Observed Effect Concentration" (NOEC):≥100 mg/l

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.