Reaction mass of chromium (+ 3) hydroxide sulfate and sodium sulfate

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:

2009-11-30 - 2010-07-08 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 'Freshwater Algae and Cyanobacteria, Growth inhibition test' (2009)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test' (2006)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.0032, 0.01, 0.032, 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L and a control
- Sampling method: In order to avoid an impairment of the test system, an additional replicate was used for analysis at the beginning of the test. Chemical analysis and pH measurement at the end of the test were performed using replicate 1 of the test.
Additional replicates were used for quantitative examination of samples (Chromium determination) after 0, 24, 48 and 72 h. In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, test flasks at the test concentration of 1.0 mg/L, without algae, were run in parallel to the geometric series of test concentrations.
- Sample storage conditions before analysis: Routinely, the samples are analysed immediately. Only in exceptional cases, they were stored overnight deeply frozen and protected from light.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 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.
- Controls: Consisting of nutritient medium only.
- Test concentration separation factor: 3.2

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus (formerly Scenedesmus subspicatus)
- Strain: Strain No. 86.81 SAG
- Source (laboratory, culture collection): Obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Age of inoculum (at test initiation): 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.
- Method of cultivation: 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.

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.

Hardness:

Water hardness of the final nutrient medium was 1.3°dH, corresponding to 22.5 mg/L CaCO3

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: 0.0032, 0.01, 0.032, 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L and a control
measured initial concentrations: 1.28, 4.01, 8.89, 15.47, 52.49, 173.33, 327.53, 313.72, 637.73 and 848.61 µg/L and a control

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Type (delete if not applicable): closed with cotton stoppers only
- Material, size, headspace, fill volume: glass
- Initial cells density: 5000 cells per millilitre
- Control end cells density: 513330 cells per millilitre
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Other: Three additional replicates each, per test concentration and control for analytical purpose and one additional replicate of the test concentration 1.0 mg/L without algae.

GROWTH MEDIUM
- Standard medium used: yes (nutrient medium according to BRINGMANN & KÜHN (1977))

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Please find a table providing the detailled composition of the nutritient medium of pre-cultures and test cultures attached as background material.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: The pH of the concentrated nutrient medium was measured to be pH 8.1 and was adjusted to pH 6.0. During the test, the pH value of the test media was adjusted every day to pH 6.0 ± 0.4 in all replicates.
- Photoperiod: continuous uniform illuminationwith a spectral range of 400 to 700 nm.
- Light intensity and quality: 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.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r], respectively, of the algal population after 24, 48 and 72 hours of exposure.
- Determination of cell concentrations: 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.
- Other: The flasks were cultured in a light chambre.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study : Two range-finding (non-GLP) tests preceded the main test and provided information about the range of concentrations which were used in the main test. The following nominal concentrations of the test item were tested in the range-finding tests: 0.01 mg/L, 0.1 mg/L, 1.0 mg/L, 10 mg/L and 100 mg/L (first range-finding test); 0.001 mg/L, 0.01 mg/L, 0.1 mg/L and 1.0 mg/L (second range-finding test).

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

76.1 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% confidence limits: 20.9 - 282.7

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.29 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% confidence limits: 0.00 - 2.5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.01 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 848.6 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: not determinable

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11.7 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 0.06 - 46.3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.01 µg/L

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:

yield

Remarks on result:

other: 95% confidence limits: 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:

yield

Remarks on result:

other: 95% confidence limits: 0.00 - 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:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 148.1 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Remarks:

chromium

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: not determinable

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: 95% confidence limits: 0.010 - 8.1

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:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not stated

Results with reference substance (positive control):

not stated

Reported statistics and error estimates:

All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 19.02.2009). For the calculations all algae counts were divided by a factor of 10000. The NOEC and LOEC were determined according to Williams Multiple Sequential t-test Procedure. Effective concentrations were calculated by Probit analysis.

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to 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) on the registered substance, without deviations which may have an impact on the validity of the study. Thus, the results were obtained via a scientifically reasonable method and the obtained results can be considered as reliable to assess the toxicity of the test substance to aquatic algae.
In a static test the freshwater green algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) was exposed to the test substance at the concentrations of nominal 0.0032, 0.01, 0.032, 0.1, 0.32, 1.0, 3.2, 32 and 100 mg/L and a control for a period of 72 hours under defined conditions. Using ICP-MS analysis initial mean measured concentrations of 1.28, 4.01, 8.89, 15.47, 52.49, 173.33, 327.53, 313.72, 637.73 and 848.61 µg Cr/L (dissolved chromium) were determined.
Based on the obtained results the 72 hour ErC50 of was determined to be > 848.6 µg/L based on initial measured concentrations of the test material, corresponding to 148.1 µg/L based on the initial mean measured concentration of dissolved Chromium. The 72 hour ErC10 was determined to be 11.7 and 2.0 µg/L for initial mean measured concentrations of the test material and dissolved Chromium, respectively, and the 72 hour NOErC was determined to be 4.01 and 0.70 µg/L.

Executive summary:

The study was performed to assess the adverse effects of the registered substance on yield (= biomass at time t minus initial biomass) and the growth rate (= rate of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.The study was conducted under GLP 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 forTesting 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 of Chromium (III) hydroxide sulphate dissolved in water and a control. The pH value was adjusted every 24 hours to pH 6.0 ± 0.4 during the whole study. The results are expressed in terms of measured initial concentrations. This is explicitly recommended in the guideline in those cases, where there is a decrease inconcentration 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 for1.0 mg/L and 10 mg/L).

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration.

The following values were determined:

Based on measured initial concentrations of the test material:

ErC50 (72h) > 848.6 µg/L (95% confidence limits: not determinable)

ErC10 (72h) = 11.7 µg/L (95% confidence limits: 0.06 – 46.3 µg/L)

NOErC (72h) = 4.01 µg/L

EyC50 (72h) = 76.1 µg/L (95% confidence limits: 20.9 – 282.7 µg/L)

EyC10 (72h) = 0.29 µg/L (95% confidence limits: 0.00 – 2.5 µg/L)

NOEyC (72h) = 4.01 µg/L

 

Based on measured initial concentrations of dissolved Chromium:

ErC50 (72h) > 148.1 µg/L (95% confidence limits: not determinable)

ErC10 (72h) = 2.0 µg/L (95% confidence limits: 0.010 – 8.1 µg/L)

NOErC (72h) = 0.70 µg/L

EyC50 (72h) = 13.3 µg/L (95% confidence limits: 20.9 – 282.7 µg/L)

EyC10 (72h) = 0.051 µg/L (95% confidence limits: 0.00 – 2.5 µg/L)

NOEyC (72h) = 0.70 µg/L

A 72 h ErC50 (growth rate) > 848.6 µg/L and an ErC10 (growth rate) of 11.7 µg/L were determined for Chromium (III) hydroxide sulphate, corresponding to an ErC50 > 148.1 and ErC10 of 2.0 µg/L dissolved chromium (III) (measured, initial), respectively.

Description of key information

Key_Toxicity to aquatic algae and cyanobacteria: Desmodesmus subspicatus ErC50 (72h) > 849 µg/L (nominal) and > 148 µg Cr/L (initial mean measured), ErC10 (72h) = 12 µg/L (nominal) and 2 µg Cr/L (initial mean measured) (static, freshwater, OECD 201 and EU Method C.3, GLP)

Key value for chemical safety assessment

EC50 for freshwater algae:

148 µg/L

EC10 or NOEC for freshwater algae:

2 µg/L

Additional information

The study was performed to assess the adverse effects of the registered substance on yield (= biomass at time t minus initial biomass) and the growth rate (= rate of increase in cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.The study was conducted under GLP in accordance with Commission Regulation (EC)No 761/2009 amending Regulation No 440/2008, Method C.3 'Freshwater Algae andCyanobacteria, 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 of Chromium (III) hydroxide sulphate dissolved in water and a control. The pH value was adjusted every 24 hours to pH 6.0 ± 0.4 during the whole study. The results are expressed in terms of measured initial concentrations. This is explicitly recommended in the guideline in those cases, where there is a decrease inconcentration 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 for1.0 mg/L and 10 mg/L).

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration.

The following values were determined:

Based on measured initial concentrations of the test material:

ErC50 (72h) > 848.6 µg/L (95% confidence limits: not determinable)

ErC10 (72h) = 11.7 µg/L (95% confidence limits: 0.06 – 46.3 µg/L)

NOErC (72h) = 4.01 µg/L

EyC50 (72h) = 76.1 µg/L (95% confidence limits: 20.9 – 282.7 µg/L)

EyC10 (72h) = 0.29 µg/L (95% confidence limits: 0.00 – 2.5 µg/L)

NOEyC (72h) = 4.01 µg/L

 

Based on measured initial concentrations of dissolved Chromium:

ErC50 (72h) > 148.1 µg/L (95% confidence limits: not determinable)

ErC10 (72h) = 2.0 µg/L (95% confidence limits: 0.010 – 8.1 µg/L)

NOErC (72h) = 0.70 µg/L

EyC50 (72h) = 13.3 µg/L (95% confidence limits: 20.9 – 282.7 µg/L)

EyC10 (72h) = 0.051 µg/L (95% confidence limits: 0.00 – 2.5 µg/L)

NOEyC (72h) = 0.70 µg/L

A 72 h ErC50 (growth rate) > 848.6 µg/L and an ErC10 (growth rate) of 11.7 µg/L were determined for Chromium (III) hydroxide sulphate, corresponding to an ErC50 > 148.1 and ErC10 of 2.0 µg/L dissolved chromium (III) (measured, initial), respectively.