Chromium trioxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978 to 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The EU RAR summarises results from a number of algal studies which, individually, have limitations regarding study design and reliability. However, the results of these studies when taken as a whole, provide an adequate assessment of the toxicity to algae. The data are considered to fulfil the criteria laid down in Annex XI to Regulation 1907/2006: adequate for classification and labelling, adequate coverage of key parameters (growth inhibition), exposure duration comparable or longer that Article 13(3) methods (72 hours), adequate documentation provided: EU RAR, of which parts have been copied to this endpoint record. Reliability for endpoint also increased based on test results from several species.

Qualifier:

no guideline followed

Guideline:

other: range of studies included

Principles of method if other than guideline:

Several studies, collectively, provide a weight-of-evidence to support toxicity to algae

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

Various sampling procedures based on multiple studies

Vehicle:

no

Details on test solutions:

Various test media prepared based on multiple studies in freshwater and brackish water

Test organisms (species):

other: Range of species tested

Test type:

static

Limit test:

no

Remarks on exposure duration:

Range used from several studies

Post exposure observation period:

None reported

Hardness:

Range used from several studies

Test temperature:

Range used from several studies

pH:

Range used from several studies

Dissolved oxygen:

Not applicable

Salinity:

Range used from several studies

Nominal and measured concentrations:

Range used from several studies

Details on test conditions:

Range used from several studies

Reference substance (positive control):

no

Details on results:

Potassium dichromate is recommended as a reference substance in the algal inhibition test (Method C.3; EEC, 1992). A ring test involving 16 laboratories determined the mean 72h-EC50 values for Scenedesmus subspicatus and Selenastrum capricornutum. The mean and range of the
values obtained (for the two species combined) are shown below on both a K2Cr2O7- concentration basis (EEC, 1992) and also converted to the equivalent chromium concentration.

Endpoint mean value range:
EC50(growth rate) 0.84 mg K2Cr2O7/l 0.6-1.03 mg K2Cr2O7/l =0.30 mg Cr/l =0.21-0.36 mg Cr/l
EC50(biomass) 0.53 mg K2Cr2O7/l 0.20-0.75 mg K2Cr2O7/l =0.19 mg Cr/l =0.071-0.26 mg Cr/l.

Most of the algal toxicity data have been generated with potassium dichromate. Where comparison is possible, the toxicity of sodium chromate or sodium dichromate, when expressed on a total chromium basis, does not appear to be significantly different from that of potassium dichromate. This is as would be expected if the equilibria between the chromate and dichromate anions are established in the test medium. Little information is available for ammonium dichromate and chromic acid, but it would be expected that their toxicity would be similar to that of the other chromates/dichromates, when expressed on a total chromium concentration basis.

The available EC50 values for algae and plants range from 0.13 to 4.6 mg/l Cr; NOEC values are in the range 0.01 to 0.64 mg/l Cr.

With marine algae, salinity and sulphate ion concentration have been shown to be important factors in determining the toxicity of chromium (VI). The toxicity of chromium (VI) is generally highest at low salinities (<2‰) and low sulphate ion concentrations (Riedel, 1984 and 1985;Frey et al., 1983). At higher salinities, marine alga appears to be of similar or slightly lower sensitivity as freshwater algae.

Riedel (1985) studied the uptake of chromium (VI) (as potassium dichromate) by the marine diatom Thalassiosira pseudonana at low salinities (0.32-3.2‰) and sulphate ion concentrations. It was found that the rate of uptake of chromium (VI) was approximately linear with time, proportional to the aqueous chromium (VI) concentration and inversely proportional to the aqueous sulphate concentration. The concentration of chromium (VI) that inhibited cell growth also inhibited sulphate uptake.

The results of the algal toxicity studies evaluated in the EU RAR are summarised below.

Species	Endpoint: growth (g) biomass (b)	Value (mg/L)	Reference
Algae - freshwater
Chlamydomonas sp	10 d NOEC (g)c	0.5	Cairns Jr. et al. (1978)
Chlorella vulgaris	72 h IC50 (g)	0.47	Jouany et al. (1982)
Chlorella pyrenoidosa	96 h NOEC (b)	0.1	Meisch and Schmitt-Beckmann (1979)
Chlorella sp (wild)	96 h NOEC (b)	0.1	Meisch and Schmitt-Beckmann (1979)
Lyngbya (blue-green)	18 d NOEC (g)c	0.1	Cairns Jr. et al. (1978)
Microcystis aeruginosa (bluegreen)	96 h NOEC (g)	0.35	Sloof and Canton (1983)
Scenedesmus pannonicus	96 h NOEC	0.11	Sloof and Canton (1983)
Scenedesmus subspicatus	72 h EC50 (g)	4.6	Kuhn and Pattard (1990)
	72 h EC10 (g)	0.64
	72 h EC50 (b)	0.13
	72 h EC10 (b)	0.032
Selenastrum capricornutum	72 h IC50 (g)	0.99	Nyholm (1991)
	72 h IC10 (g)	0.11
	96 h EC50 (b)	0.217	Greene et al. (1988)
	72 h EC50 (g)	0.233	Christensen et al. (1983) Christensen and Nyholm (1984)
	72 h EC10 (g)	0.01
Algae- brackish water
Thalassiosira pseudonana	EC50	0.341	-l Riedel(1984)

a NOEC calculated as LOEC/2 from paper, effect of 14%; (b) - biomass; (g) – growth

b sodium chromate tested; all others used potassium dichromate. All concentrations as Cr.

c duration of test too long for inclusion in PNEC derivation.

d study rated as IIIb

Validity criteria fulfilled:

yes

Conclusions:

Based on a review of existing algal data from the EU RAR, an acceptable assessment of toxicity was achieved.

Executive summary:

From the EU RAR, most of the algal toxicity data have been generated with potassium dichromate. Where comparison is possible, the toxicity of sodium chromate or sodium dichromate, when expressed on a total chromium basis, does not appear to be significantly different from that of potassium dichromate. This is as would be expected if the equilibria between the chromate and dichromate

anions are established in the test medium. Little information is available for ammonium dichromate and chromic acid, but it would be expected that their toxicity would be similar to that of the other chromates/dichromates, when expressed on a total chromium concentration basis.

The available EC50 values for algae range from 0.13 to 4.6 mg/l Cr; NOEC values are in the range 0.01 to 0.64 mg/l Cr. With marine algae, salinity and sulphate ion concentration have been shown to be important factors in determining the toxicity of chromium (VI). The toxicity of chromium (VI) is generally highest at low salinities (<2‰) and low sulphate ion concentrations (Riedel, 1984 and 1985; Frey et al., 1983). At higher salinities, marine alga appears to be of similar or slightly lower sensitivity as freshwater algae.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

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:

other: DIN 38 412

Version / remarks:

Part 9

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In order to produce the test solutions, water-soluble substances were quantitatively dissolved to produce an optically clear stock solution in 800 mL double-distilled water using magnetic stirrers (maximum 24 h). Optically unclear solutions were filtered through fibre-glass filters (Selecta No. 6) and the filtrate was quantified chemically. Before beginning the test, the stock solutions were adjusted to pH 8.0 +/- 0.3. From the test stock solution and from sterile double distilled water as the diluent, dilution series with various volume ratios were set up.
- Controls: yes, prepared from dilution water, nutrient solution and algal suspension
- Test concentration separation factor: Dilution steps with a dilution series of 1:2 (f=2) each had one part of test substance solution in 2^0-2^8 volumes of mixture.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain no.: 8681 SAG
- Source (laboratory, culture collection): in-houselaboratory culture maintained over a number of decades in submerged culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: For the maintainance of the green algal species in a submerged culture, sterilised 100 mL Erlenmeyer flasks were filled with 20 mL nutrient solution (for stock cultures) and inoculated with 2 mL cell suspension taken from a 10-day old stock culture. The inoculated flasks were placed on a white surface, protected from daylight and exposed to constant lighting from two parallel fluorescent Osram 40 W/30 tubes (distance from each tube 60 cm: irradiance E0, Sy = 24.9 W/m^2) at 24 +/- 1°C and relative humidity of 50%. To maintain the test strain, fresh stock cultures were prepared at 10-day intervals.

ACCLIMATION
- Acclimation period: The cultivation of the preliminary cultures was undertaken 3 days prior to the preparation of the test solution. 50 mL nutrient solution (for test cultures) were filled into 300 mL Erlenmeyer flasks and inoculated with cell suspension from 7-day-old stock cultures. The cell concentration in the preliminary culture flasks must amount to 104.ml-L This was to ensure that the culture was still in a process of logarithmic growth after 72 h.
- Culturing media and conditions (same as test or not): Light and temperature conditions of the preliminary culture corresponded to those for the test preparation. The composition of the nutrient solution used for culturing is presented in section "Any other information on materials and methods incl tables".

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

not specified

pH:

between 8.0 and 9.3

Dissolved oxygen:

not specified

Nominal and measured concentrations:

nominal: 0.08 - 10 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks with metal caps
- Type: closed (sterila)
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flasks filled with 250 mL test solution
- Initial cells density: 10^4 mL^-1
- Control end cells density: 1.8x10^6 mL^-1
- No. of vessels per concentration (replicates): 8 (thus, on each measurement day two flasks from each concentration were available for measurement)
- No. of vessels per control (replicates): 8 (thus, on each measurement day two flasks were available for measurement)

GROWTH MEDIUM
- Standard medium used: Yes, nutrient solution for the test preparation according to DIN Test Procedure L9.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Nutritient solution and destilled water. The composition and information on the preparation of the test medium is presented in section "Any other information on materials and methods incl. tables".

OTHER TEST CONDITIONS
- Adjustment of pH: yes, at test start in stock solutions of test substance to pH 8.0 +/- 0.3
- Photoperiod: constant lighting
- Light intensity and quality: The light was provided by Osram L 25/40 fluorescent tubes; each of the positions for the test solutions had an irradiance E=17.0 W/m^2.
- Other: The control and test preparations were shaken daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: via extinction measurement of the monochromatic radiation (578 nm); biomass was determined by measurement of optical density (measurement of turbidity)
- Equipment: Eppendorf digital photometer 6115 S with a facility to measure turbidity, filter 578 nm
- Intervals: Measurements were made at the beginning of the test and after 24, 48, 72 and 96 hours of test duration.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.38 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.09 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: no

Reported statistics and error estimates:

The data were evaluated in line with the DIN Standard. Growth curves were etsablished for each tested concentration of the test materail and for the control. The area under the growth curves (integral of the biomass with time) was calculated according to the given equation and from this the "percentage inhibition of cell multiplication (H_B) during the test for each tested concentration level on the basis of a comparison of the biomass formed under the influence of the substance (B_T) with the biomass in the control (B_K)" was calculated. The average growth rate for cultures showing exponential growth was calculated, the basis being the time between the beginning of the test and 72 h later. On this basis the growth-related inhibition (Hµ) was calculated from the difference between the growth rates (µ_k - µ_T) and the growth rate µ_K in the control. The tested concentrations were assigned to the respective inhibition values in the probability paper. The regression line was determined and from this the desired values EBC10 and EBC50 and/or EµC10 and EµC50 could be read.

Validity criteria fulfilled:

yes

Conclusions:

A growth inhibition study was conducted with the freshwater green-algal species Scenedesmus subspicatus to determine the toxicity of potassium dichromate to aquatic algae and cyanobacteria. The test was conducted according to Standard Protocol DIN 38 412 (Part 9), the procedure is well described and the validity criteria were met. Hence, the results can be considered to be reliable and acceptable to assess the hazard of the registered substance to aquatic algae.
During the test an initial cell density of 10^5 algal cells per mL was exposed to several concentrations of the test material under sterile static test conditions for a period of 96 hours. A control group was running in parallel. The biomass was determined daily by measurement of the optical density for each test vessel and the obtained results were used to determine the effect on algal growth compared to the control cultures. Based on the obtained results the 72h EC10 and the 72h EC50 value for the parameter growth rate (EµC50) were determined to be 1.8 and 10 mg/L, respectively. The 72 EC10 and the 72h EC50 for the parameter biomass (EbC50) were determined to be 0.09 and 0.38 mg/L, respectively.

Executive summary:

In the Scenedesmus cell multiplication inhibition test, 68 potentially hazardous substances were examined to determine the effect concentrations (EC). One of them was potassium dichromate. The tests were conducted in accordance with the test procedure DIN 38 412, Part 9 (draft standard). The green alga Scenedesmus subspicatus CHODAT was cultivated as the test organism. During the test an initial cell density of 10^5 algal cells per mL was exposed to several concentrations of the test material under sterile static test conditions for a period of 96 hours. A control group was running in parallel. The biomass was determined daily by measurement of the optical density for each test vessel and the obtained results were used to determine the effect on algal growth compared to the control cultures.

Based on the obtained results the 72h EC10 and the 72h EC50 value for the parameter growth rate (EµC50) were determined to be 1.8 and 10 mg/L, respectively. The 72h EC10 and the 72h EC50 for the parameter biomass (EbC50) were determined to be 0.09 and 0.38 mg/L, respectively.

In a comparison of the concentrations of the E_BC₁₀ for Scenedesmus with those of the corresponding NOEC for Daphnia magna - bearing in mind that the values are not based on the same measurement - it was shown that both endpoints are quite similar for potassium dichromate and therefore comparable. However, for risk assessment the ECx values for the parameter growth rate will be used as key results.

Description of key information

 1) Key_ Toxicity to aquatic algae and cyanobacteria by summary of data from European Union Risk Assessment Report: chromium trioxide, sodium chromate, sodium dichromate, ammonium dichromate and potassium dichromate; 3rd. Priority List; Volume 53: range for EC50: 0.13 to 4.6 mg/L Cr; range for NOEC: 0.01 to 0.64 mg/L Cr for several species from various studies with different test durations ranging from 3 to 18 days.

2) Key_Toxicity to aquatic algae and cyanobacteria: 72h-EC50 (growth rate): 10 mg/L K₂Cr₂O₇; 72h-EC10 (grwoth rate): 1.8 mg/L K₂Cr₂O₇; for Desmodesmus subspicatus according to DIN 38 412 (comparable to OECD TG 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.13 mg/L

EC10 or NOEC for freshwater algae:

0.01 mg/L

Additional information

1) From the EU RAR, most of the algal toxicity data have been generated with potassium dichromate. The available EC50 values for algae range from 0.13 to 4.6 mg/L Cr; NOEC values are in the range 0.01 to 0.64 mg/L Cr. With marine algae, salinity and sulphate ion concentration have been shown to be important factors in determining the toxicity of chromium (VI). The toxicity of chromium (VI) is generally highest at low salinities (<2‰) and low sulphate ion concentrations. At higher salinities, marine alga appears to be of similar or slightly lower sensitivity as freshwater algae.

2) In the Scenedesmus cell multiplication inhibition test, 68 potentially hazardous substances were examined to determine the effect concentrations (EC). One of them was potassium dichromate. The tests were conducted in accordance with the test procedure DIN 38 412, Part 9 (draft standard). The green alga Scenedesmus subspicatus CHODAT was used as test organism and exposed to several concentrations of the test material under sterile static test conditions for a period of 96 hours. A control group was running in parallel. Based on the obtained results the 72h EC10 and the 72h EC50 value for the parameter growth rate (EµC₅₀) were determined to be 1.8 and 10 mg/L, respectively. The 72h EC10 and the 72h EC50 for the parameter biomass (E_BC₅₀) were determined to be 0.09 and 0.38 mg/L, respectively.

In a comparison of the concentrations of the E_BC₁₀ for Scenedesmus with those of the corresponding NOEC for Daphnia magna - bearing in mind that the values are not based on the same measurement - it was shown that both endpoints are quite similar for potassium dichromate and therefore comparable. However, for risk assessment the ECx values for the parameter growth rate will be used as key results.