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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP guideline-compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of chromium (+ 3) hydroxide sulfate and sodium sulfate
EC Number:
914-129-3
Cas Number:
12336-95-7
Molecular formula:
CrHO5S
IUPAC Name:
Reaction mass of chromium (+ 3) hydroxide sulfate and sodium sulfate
Details on test material:
Chromium (III) hydroxide sulphate, batch Südafrika.
Specification - 25.5% Cr2O3 in purity (chrome content expressed as Cr2O3) and desdcribed as a green powder. The internal sampe number was 105779.
Chromium hydroxide sulphate is a composite complex multi-constituent compound which represents the basic salt of chromium sulphate. It is represented by the formula Cr2(OH)x(SO4)y, where x and y can vary to an extent depending on the manufacturing process. Technical grade material contains sodium sulphate, which is a side product formed from the adjustment of the ‘basicity’ (the proportion of OH). Technical grade material also contains water that cannot be further removed without causing deterioration of the sulphate complex.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The stability of the test concentrations during exposure were examined by chemical analysis (Chromium determination) at 0, 24, 48 and 72 hours of the test item concentrations 0.01 mg/L, 0.1 mg/L, 1.0 mg/L, 10 mg/L and 100 mg/L. The samples from the biological test were directly analysed after membrane filtration by ICP-MS detection.

Test solutions

Vehicle:
no
Details on test solutions:
A stock solution was prepared to give the desired series of test concentrations. To achieve this 375.2 mg of the test item were added to 3 litres of deionised water. The pH was measured to be pH 4.3 and was adjusted to pH 6.0 . After pH adjustment, precipitation of test item was observed. The undissolved particles of the stock solution were not removed. To produce the different test item concentrations appropriate amounts of the prepared stock solution were taken under constant stirring and were diluted with deionised water to a volume of 800 mL.
The pH of the concentrated nutrient medium was measured to be pH 8.1 and was adjusted to pH 6.0. 100 mL of the nutrient medium and 100 mL of algal inoculum with a cell density of 5000 cells/mL were added to each test item concentration resulting in the final nominal concentrations. For each test item concentration and control 6 replicates and the test concentrations were 3 replicates prepared.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Desmodesmus subspicatus (formerly Scenedesmus subspicatus) Strain No. 86.81 SAG, obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
Exponentially-growing stock cultures were maintained in the test facility under constant temperature conditions (21-24°C with a maximum fluctuation of +/- 2°C) at a light intensity in the range 60 – 120 µE. x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The nutrient medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell- counter, Sysmex F300, Digitana.
Pre-cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different nutrient medium. The algal inocula for a test were taken from an exponentially-growing pre-culture and were mixed with the nutrient medium to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
There was no post exposure observation period.

Test conditions

Hardness:
Not applicable
Test temperature:
21°C to 24°C
pH:
The pH was measured at the beginning of the test and after every 24 hours thereafter of exposure. After 24 and 48 hours the pH was adjusted to pH 6.0 ± 0.4
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations of 0.0032, 0.01, 0.032, 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.
Details on test conditions:
The test vessels were 300 mL Erlenmeyer flasks with cotton stoppers. The flasks were cultured in a light chamber in which a temperature in the range 21°C to 24°C was maintained at +/- 2°C, and continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature was measured and recorded daily in a water filled flask which was incubated under the same conditions as the test flasks. At the average of the test solutions, a light intensity in the range 60 to 120 µE. x m-2 x s-1, or an equivalent range of 4000 to 8000 lx, was used. Light intensity was checked before start of the study.
Cell densities were measured at 24 hour intervals in a microcell counter (Sysmex F300, Digitana) by taking small aliquots from each test flask for measurements, which were not replaced.
There were 3 replicates per concentration and 6 replicates per vehicle control, and a true negative control consisting of the test substance at 1.0 mg/L without algae.
Additional replicates were used to determine Chromium precipitated and/or taken up in the algae biomass. After 48 and 72 hours of exposure the replicates were centrifuged for 20 minutes at 9000 rpm and 20 °C. Afterward the supernatant and the “algae pellet” were analysed for the Chromium content.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
76.14 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 20.93 - 282.65
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.29 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 0.0 - 2.54
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.01 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 848.61 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
11.66 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 0.06 - 46.33
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.01
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
13.3 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Remarks:
chromium
Basis for effect:
biomass
Remarks on result:
other: 3.7 - 49.3
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.051 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Remarks:
chromium
Basis for effect:
biomass
Remarks on result:
other: 0.0 - 0.44
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.7 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Remarks:
chromium
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 148.1 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Remarks:
chromium
Basis for effect:
growth rate
Remarks on result:
other: 0.01 - 8.1
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.7
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Remarks:
chromium
Basis for effect:
growth rate
Details on results:
The results are expressed in terms of measured initial concentrations. This is explicitly recommended in the guideline in those cases, where there is adecrease in concentration of test item over time, which is not accompanied by a decrease in toxicity over time. This was the case in the present test.

Effective concentrations of dissolved Chromium ranged from 0.85-40.1% of nominal Chromium values at 0 hours, from 0.017-37.2% of nominal Chromium values at 24 hours, from 0.019-34.4% of nominal Chromium values at 48 hours and from 0.033-17.2% of nominal Chromium values at 72 hours, respectively. The test concentrations for which no analytics were performed (0.0032 mg/L, 0.032 mg/L, 0.32 mg/L, 3.2 mg/L and 32 mg/L) were calculated on basis of the analytically analysed proximate (i.e. next nearest) concentrations (e.g. average of 0.01 mg/L and 0.1 mg/L for 1.0 mg/L and 10 mg/L).
Results with reference substance (positive control):
A positive control was not included.
Reported statistics and error estimates:
The NOEC and LOEC were determined according to Williams Multiple Sequential t-test Procedure. Effective concentrations were calculated by Probit analysis.

Any other information on results incl. tables

Results of the chromium analysis are presented below:

Test item conc. (mg/L)

Chromium conc. (µg/L)

Dissolved chromium (ca 17.5% of the test substance)

0 hours

24 hours

48 hours

72 hours

Control

-

0.8 / 0.8
mean – 0.8

0.3 / 0.4
mean - 0.35

0.2 / 0.2
mean – 0.2

1.31/ 1.31
mean – 1.31

0.0032

0.56

0.22

0.21

0.19

0.10

0.01

1.75

0.7 / 0.7

mean – 0.7

0.6 / 0.7
mean – 0.65

0.6 / 0.6
mean – 0.6

0.3 /0.3
mean – 0.3

0.032

5.58

1.55

1.65

1.47

0.59

0.1

17.45

2.7 / 2.7
mean – 2.7

3.8 / 3.8
mean – 3.8

3.2 / 3.2
mean – 3.2

0.7 / 0.7

mean – 0.7

0.32

55.85

9.16

8.31

6.46

1.56

1.0

174.5

30.5 / 30.0
mean – 30.25

13.9 / 14.0
mean – 13.95

8.3 / 8.5
mean – 8.4

2.8 / 2.7
mean 2.75

3.2

558.5

57.16

24.77

16.5

5.14

10

1745

55.7 / 53.8
mean – 54.75

15.6 / 15.0
mean – 15.3

19.5 / 18.7
mean 19.1

4.6 / 4.7
mean 4.65

32

5585

111.3

24.95

31.1

8.35

100

17452

150.5 / 145.7

mean – 148.1

3.0 / 2.9

mean – 2.95

3.4 / 3.4
mean – 3.4

5.7 / 5.7
mean – 5.7

1.0 (no algae)

174.5

15.1 / 15.0
mean 15.05

8.6 / 8.6

mean – 8.6

17.7 / 18.4
mean – 18.05

5.9 / 6.1

Mean – 6.0

1.0 (algal pellet)

174.5

-

-

90.22/87.52

mean – 88.852

1002/ 1002

mean - 1002

1.0 (supernatant)

174.5

-

-

13.22/ 12.82

Mean – 13.02

4.22/ 4.12
mean – 4.152

1the slightly elevated level of Chromium in the control after 72 h could not be explained, but is not relevant to the overall result of the study as all validity criteria were fulfilled.
2total chromium content

The concentrations of chromium (III) hydroxide sulphate, based on conversion from the measured chromium concentrations are presented below:

Calculated chromium (III) hydroxide sulphate concentrations

Test item conc. (mg/L)

Mean chromium (III) hydroxide sulphate (µg/L)

0 hours

24 hours

48 hours

72 hours

Control

-

-

-

-

0.0032

1.28

1.19

1.10

0.55

0.01

4.01

3.72

3.44

1.72

0.032

8.89

9.44

8.43

3.39

0.1

15.47

21.77

18.34

4.01

0.32

52.49

47.63

37.04

8.94

1.0

173.33

79.93

48.13

15.76

3.2

327.53

141.92

94.52

29.48

10

313.72

87.67

109.44

26.64

32

637.73

142.97

178.23

47.86

100

848.61

16.90

19.48

32.66

1.0 (no algae)

86.24

49.28

103.43

34.38

1.0 (algal pellet)

-

-

509.11

574.72

1.0 (supernatant)

-

-

74.49

23.78

The conversion is based on the assumption that 1 mg/L chromium is equivalent to 5.73 mg/L of chromium (III) hydroxide sulphate.

Mean cell number measured during the study

Initial measured conc. (µg/L)

Mean cell number

0 hours

24 hours

48 hours

72 hours

Control

0.5

2.50

16.5

51.33

1.28

0.5

2.667

16.33

49.33

4.01

0.5

2.667

15.33

45.33

8.89

0.5

2.667

15.33

26.667

15.47

0.5

2.333

15.0

25.0

52.49

0.5

2.667

15.33

29.333

173.33

0.5

2.333

12.33

22.333

327.53

0.5

2.333

10.33

24.667

313.72

0.5

2.667

8.667

20.667

637.73

0.5

2.333

3.667

11.0

848.61

0.5

3.0

8.333

18.0

 

 

Calculated yield during the study

Initial measured conc. (µg/L)

Yield

0 hours

24 hours

48 hours

72 hours

% decrease after 72 hours

Control

0.0

2.0

16.0

50.833

-

1.28

0.0

2.167

15.833

48.833

3.9

4.01

0.0

2.167

14.833

44.833

11.8

8.89

0.0

2.167

14.833

26.167

48.5

15.47

0.0

1.833

14.5

24.5

51.8

52.49

0.0

2.167

14.833

28.833

43.3

173.33

0.0

1.833

11.833

21.833

57.0

327.53

0.0

1.833

9.833

24.167

52.5

313.72

0.0

2.167

8.167

20.167

60.3

637.73

0.0

1.833

3.167

10.5

79.3

848.61

0.0

2.5

7.833

17.5

65.6

 

Calculated specific growth rate during the study

Initial measured conc. (µg/L)

Mean specific growth rate

0 hours

24 hours

48 hours

72 hours

% decrease after 72 hours

Control

0.0

1.589

1.746

1.541

-

1.28

0.0

1.657

1.742

1.530

0.7

4.01

0.0

1.657

1.711

1.501

2.6

8.89

0.0

1.657

1.693

1.289

16.4

15.47

0.0

1.521

1.701

1.287

16.5

52.49

0.0

1.657

1.711

1.342

12.9

173.33

0.0

1.521

1.601

1.263

18.1

327.53

0.0

1.521

1.504

1.274

17.3

313.72

0.0

1.657

1.406

1.234

19.9

637.73

0.0

1.521

0.992

1.024

33.5

848.61

0.0

1.792

1.401

1.174

23.8

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 72 hour ErC50of chromium (III) hydroxide sulphate was > 848.6 µg/L, the corresponding no-observed effect concentration (NOEC) was 4.01 µg/L. The 72 hour EyC50 of chromium (III) hydroxide sulphate was 76.1 µg/L, the corresponding NOEC was 4.01 µg/L.

This substance doesn't need to be classified in the environment, though the EC 50 value is more than 848.6 ug/L (< 1 mg/L). The solubility of this substance in the enviroment (PH neutral) is 43 ug/L, which is far below the EC 50 value. Therefore, the calissification of this substance in the environment is not necessary.



Executive summary:

A study was performed to assess the effects of Chromium (III) hydroxide sulphate on the aglae Desmodesmus subspicatus. The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Algae and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a range of concentrations, nominally 0.0032, 0.01, 0.032, 0.1, 0.32, 1.0, 3.2, 32 and 100 mg/L. Initial measured concentrations were 1.28, 4.01, 8.89, 15.47, 52.49, 173.33, 327.53, 313.72, 637.73 and 848.61 µg/L. Measured values in the test media declined at subsequent analytical sampling intervals.  The results of the study are based on the initial measured concentrations; this is recommended in the guideline in those cases, where there is a decrease in concentration of test item over time, which is not accompanied by a decrease in toxicity over time.

 

The 72 hour ErC50of chromium (III) hydroxide sulphate was > 848.6 µg/L, the corresponding no-observed effect concentration (NOEC) was 4.01 µg/L. The 72 hour EyC50of chromium (III) hydroxide sulphate was 76.1 µg/L, the corresponding NOEC was 4.01 µg/L

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