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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well performed study. Effect concentrations based on measured Se concentrations.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
No data
Vehicle:
no
Details on test solutions:
see test conditions
Test organisms (species):
Chlamydomonas reinhardtii
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Cumbria, United Kingdom; culture 11/32B
- Age of inoculum (at test initiation): inoculum from a preculture in the exponential growth phase
- Method of cultivation: Axenically in synthetic freshwater medium (Morlon et al., 2005a). Cultures were carried out in 250 mL Erlenmeyer flasks containing 100 mL of autoclaved medium placed in an incubator (Innova 44 R orbital shaker, New Brunswick Scientifics, Edison, NJ, USA) under continuous illumination (100 µmol photons/m²/s) provided by white fluorescent lamps (Sylvania® Grolux F 36 W) at 24±1 ◦C and with rotary agitation (120 rpm).

ACCLIMATION
- Acclimation period: no data
- Culturing media and conditions (same as test or not): yes
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
Can be calculated (2.0 mM Ca, 1.04 mM Mg)
Test temperature:
24 ± 1 °C
pH:
7
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
0, 2.5, 5, 7.5 and 10 µM (nominal)
<0.1, 2.38±0.03, 5.04±0.06, 7.2±0.2 and 9.84±0.08 µM (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel:l polycarbonate Erlenmeyer flasks containing 200 mL of test solution
- Initial cells density: 2500 cells/mL
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: synthetic freshwater medium consisting of (in mM): NH4, 9.36; Cl, 13.47; K, 1.22; PO4, 0.14; Cinorg, 50×10−3 (pCO2 = 3.5×10−4 atm); NO3, 2.92; SO4, 81.2×10−3; Mg, 1.04; Ca, 2.00; Na, 0.10; BO3, 3.01×10−3; Mn, 2.09×10−3; EDTA, 8.06×10−4; Fe, 5.92×10−4; MoO4, 3.00×10−5; Zn, 2.43×10−5; Co, 1.09×10−5 and Cu, 7.04×10−8.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic freshwater medium composed using deionized water as dilution water

OTHER TEST CONDITIONS
- Adjustment of pH: daily to pH = 7 using 0.1 M KOH
- Photoperiod: continuous illumination
- Light intensity and quality: 100 µmol photons/m²/s provided by white fluorescent lamps (Sylvania® Grolux F 36W)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Algal cell density and average cell diameter were quantified by a Coulter Z2 particle counter (1:10 dilution with ISOTONII isotonic solution, using a 100-µm orifice tube; Beckman Coulter, Roissy, France).

TEST CONCENTRATIONS
- Test concentrations: <0.1, 2.38±0.03, 5.04±0.06, 7.2±0.2 and 9.84±0.08 µM (measured)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
197 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
other: Growth (maximal cell density)
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
395 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
other: Growth (maximal cell density)
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
355 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
other: Growth (Maximal cell density)
Reported statistics and error estimates:
NOEC and LOEC values were determined using one-way ANOVA + multiple comparisons using Tukey's post-hoc test.
The add-on package drc was used to fit regression models to concentration effect relationships to estimate the 96-h EC50. Logistic and Weibull models were tested.
Conclusions:
Reliable (Klimisch 2) study on the toxicity of sodium selenate to Chlamydomonas reinhardtii. The 96-h NOEC, LOEC and EC50 were 197, 395 and 355 µg Se/L, respectively, and were based on results for growth (maximal population density).
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Tests performed according to US EPA and EU standard methods for toxicity testing. Effect concentrations based on measured Se concentrations. A K2 was assigned because the test was conducted in field water (1 µm filtered and 'uncontaminated', however, only a background concentration of Se (< 2 µg/L) was reported) and because no report was made on control requirements.
Qualifier:
according to guideline
Guideline:
other: EPA 600/491002: Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms.
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
-Frequency: At test initation and termination
Vehicle:
no
Details on test solutions:
see test conditions
Test organisms (species):
other: Dunaliella viridis
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): Laboratory culture
- Age of inoculum (at test initiation): algae were precultured for a few days until they reached the exponential growth phase

ACCLIMATION
- Acclimation period: no data
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
No data
Test temperature:
25 ± 1 °C
pH:
7.9-8.4
Dissolved oxygen:
1.8-6.0 mg/L
Salinity:
80-102 g/L
Nominal and measured concentrations:
0, 15.6, 26, 43, 72, 120 and 200 mg Se/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel:l 125-mL flasks with 50 mL of test solution
- Initial cells density: 10000 cells/mL
- No. of vessels per concentration (replicates): 16 whereof 4 replicates terminated each 24h
- No. of vessels per control (replicates): 16 whereof 4 replicates terminated each 24h

GROWTH MEDIUM
- Standard medium used: no
- Dilution water used: Great Salt Lake water passed through a 1-µm filter
- TOC = 35-49 mg/L
- DOC = 34.8-40 mg/L
- Total suspended solids = 5-18 mg/L
- Sulfate = 5800 mg/L
- Background Se < 2 µg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Great Salt Lake water, 1-µm-filtered

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Algal cell density was measured using a Hach 300 spectrophotometer (Loveland, CO, USA). The spectrophotometer was calibrated against known cell density stocks of D. viridis.

TEST CONCENTRATIONS
- Test concentrations: 0, 15.6, 26, 43, 72, 120 and 200 mg Se/L (nominal)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
11 000 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
growth rate
Remarks:
and biomass
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
18 000 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
growth rate
Remarks:
and biomass
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
45 000 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
growth rate
Remarks on result:
other: 95% CL = 36000-71000
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
32 000 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
biomass
Details on results:
biomass-based effect concentrations (area under the gowth curve):
96-h NOEC = 11000 µg/L
96-h LOEC = 18000 µg/L
96-h EC50 = 32000 µg/L
Reported statistics and error estimates:
Statistical computer package TOXIS was used to estimate the EC50 and the 95% confidence interval.
NOEC and LOEC were determined using SPSS in accordance with procedures described in the EU (1992).
Conclusions:
Reliable (Klimisch 2) study on the toxicity of sodium selenate to the saltwater alga Dunaliella viridis. Results on growth rate and biomass (area under the growth curve) were used to determine the effect concentrations. For growth rate the 96-h NOEC, LOEC and EC50 were 11000, 18000 and 45000 µg Se/L, respectively. For biomass these values were 11000, 18000 and 32000 µg Se/L, respectively.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well performed study. Effect concentrations based on measured Se concentrations.
Qualifier:
no guideline followed
Principles of method if other than guideline:
10-d experiment on the toxicity of selenium to the cyanobacterium Anabaena flos-aquae investigating the effect on growth based on chlorophyll a measurements. During the 10 days of exposure, the cyanobacteria ran through their three different growth stages (lag, exponential, and stationary).
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Samples were taken at 0, 48, 96, 144 and 240 h
Vehicle:
no
Details on test solutions:
see test conditions
Test organisms (species):
Anabaena flos-aquae
Details on test organisms:
TEST ORGANISM
- Strain: UTEX 1444
- Source (laboratory, culture collection): obtained from the university of Texas culture collection (UTEX 1444)
- Age of inoculum (at test initiation): the inoculum was taken from a culture in the log growth phase
- Method of cultivation: axenic in modified Woodshole media

ACCLIMATION
- Acclimation period: no data
- Culturing media and conditions (same as test or not): same
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
10 d
Hardness:
not reported (as in Kiffney, 1989)
Test temperature:
not reported (as in Kiffney, 1989)
pH:
not reported (as in Kiffney, 1989)
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
Control + 1.0-3.0-5.0-10.0 mg Se/L (nominal)
Control + 1.03-2.92-4.99-9.87 mg Se/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: 3-L clear glass vessels containing 2 L of water and modified Woods Hole media
- Initial cells density: 100000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: modified Woods Hole media was used - for detailed composition reference was made to Kiffney (1989)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: artificial test medium (Woods Hole) was composed using double deionized and sterilized water as dilution water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: see Kiffney (1989)
- Photoperiod: see Kiffney (1989)
- Light intensity and quality: see Kiffney (1989)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: determined by the acidification method of Strickland and Parsons (1972) and a model III Turner fluorometer

TEST CONCENTRATIONS
- Range finding study conducted to determine selenium levels which would provide a range of toxic effects (no details reported)
Duration:
10 d
Dose descriptor:
NOEC
Effect conc.:
1 030 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
other: chlorophyll a
Duration:
10 d
Dose descriptor:
LOEC
Effect conc.:
2 920 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
other: chlorophyll a
Reported statistics and error estimates:
ANOVA and Tukey's HSD test for multiple comparisons among means
Conclusions:
Reliable (Klimisch 2) study on the toxicity of selenite to the freshwater blue-green alga Anabaena flos-aquae. The 10-d NOEC and LOEC were determined to be 1030 and 2920 µg Se/L, respectively, and were based on chlorophyll a measurements.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well performed study. Effect concentrations based on measured Se concentrations.
Qualifier:
no guideline followed
Principles of method if other than guideline:
10-d experiment on the toxicity of selenium to the cyanobacterium Anabaena flos-aquae investigating the effect on growth based on chlorophyll a measurements. During the 10 days of exposure, the cyanobacteria ran through their three different growth stages (lag, exponential, and stationary).
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Samples were taken at 0, 48, 96, 144 and 240 h
Vehicle:
no
Details on test solutions:
see test conditions
Test organisms (species):
Anabaena flos-aquae
Details on test organisms:
TEST ORGANISM
- Strain: UTEX 1444
- Source (laboratory, culture collection): obtained from the university of Texas culture collection (UTEX 1444)
- Age of inoculum (at test initiation): the inoculum was taken from a culture in the log growth phase
- Method of cultivation: axenic in modified Woodshole media

ACCLIMATION
- Acclimation period: no data
- Culturing media and conditions (same as test or not): same
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
10 d
Hardness:
not reported (as in Kiffney, 1989)
Test temperature:
not reported (as in Kiffney, 1989)
pH:
not reported (as in Kiffney, 1989)
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
Control + 1.0-3.0-5.0-10.0 mg Se/L (nominal)
Control + 0.995-2.99-4.93-9.72 mg Se/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: 3-L clear glass vessels containing 2 L of water and modified Woods Hole media
- Initial cells density: 100000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: modified Woods Hole media was used - for detailed composition reference was made to Kiffney (1989)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: artificial test medium (Woods Hole) was composed using double deionized and sterilized water as dilution water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: see Kiffney (1989)
- Photoperiod: see Kiffney (1989)
- Light intensity and quality: see Kiffney (1989)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: determined by the acidification method of Strickland and Parsons (1972) and a model III Turner fluorometer

TEST CONCENTRATIONS
- Range finding study conducted to determine selenium levels which would provide a range of toxic effects (no details reported)
Duration:
10 d
Dose descriptor:
NOEC
Effect conc.:
995 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
other: chlorophyll a
Duration:
10 d
Dose descriptor:
LOEC
Effect conc.:
2 990 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
other: chlorophyll a
Reported statistics and error estimates:
ANOVA and Tukey's HSD test for multiple comparisons among means
Conclusions:
Reliable (Klimisch 2) study on the toxicity of selenate to the freshwater blue-green alga Anabaena flos-aquae. The 10-d NOEC and LOEC were determined to be 995 and 2990 µg Se/L, respectively, and were based on chlorophyll a measurements.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1992-08-18 to 1992-08-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study. Well performed. Not entirely clear whether effect concentrations were expressed as concentrations of sodium selenite or selenium. Assumed sodium selenite and recalculated to selenium.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Frequency: No data
Vehicle:
no
Details on test solutions:
see test conditions
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Selenastrum capricornutum
- Source: LISEC laboratory culture (ex. CCAP 278/4)
- Age of inoculum (at test initiation): 4 days (exponential growth phase reached after 4 days of preculturing)

ACCLIMATION
- Acclimation period: 4 days of preculturing
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
No data (hardness of algal medium recommended by OECD guideline 201)
Test temperature:
23.4-24.8 °C
pH:
8.26-9.18
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
0 (control), 10, 18, 32, 58 and 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL borosilicate glass Erlenmeyers covered with cotton wool and containing 100 mL of test medium
- Initial cells density: 10000 cells/mL
- Control end cells density: Increased by a factor of 100 within 3 days
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- In order to keep the algae in suspension and to facilitate transfer of CO2, the Erlenmeyers were continuously shaken on a laboratory shaker (Gerhardt Type LS20)

GROWTH MEDIUM
- Standard medium used: yes (according to OECD 201)

OTHER TEST CONDITIONS
- Light intensity and quality: 10000 lux (Philips TL-33 lamps)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell concentration was determined by absorbance at 0, 24, 48 and 72 hours using a Varian Superscan UV/VIS Spectrophotometer (wavelength: 720 nm, special band width: 4 nm, cuvettes with light paths of 5 cm).
- Other: At the end of the test deformations of algal cells were evaluated using a microscope at 200x magnification

TEST CONCENTRATIONS
- Range finding study: 1, 3.2, 10, 32 and 100 mg/L
- Test concentrations: 0, 10, 18, 32, 58 and 100 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4 570 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
growth rate
Remarks:
and biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
8 220 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
growth rate
Remarks:
and biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
44 240 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15 570 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
biomass
Details on results:
biomass-based effect concentrations (area under the growth curve)
- 72-h NOEC = 4570 µg/L
- 72-h LOEC = 8220 µg/L
- 72-h EC50 = 15570 µg/L
Reported statistics and error estimates:
EC50 values calculated using the logit model of Chou and Chou (1985). It is not reported which statistic test is used to determine NOEC and LOEC values.

Not entirely clear whether effect concentrations were expressed as concentrations of sodium selenite or selenium. Assumed sodium selenite and recalculated to selenium.

Validity criteria fulfilled:
yes
Conclusions:
Reliable (Klimisch 2) study on the toxicity of sodium selenite to Selenastrum capricornutum. The 72-h NOEC, LOEC and EC50 were determined using both growth rate and biomass (area under the growth curve) as endpoints. The growth rate-based NOEC, LOEC and EC50 values were 4570, 8220 and 44240 µg Se/L, respectively. Considering biomass as an endpoint the NOEC, LOEC and EC50 were 4570, 8220 and 15750 µg Se/L, respectively.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well performed study. Effect concentrations based on measured Se concentrations.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Frequency: daily
- Sampling method: Collected and filtered (0.2 µm filter PES membrane, Nalge Nunc, Rochester, NY, USA). Hydrochloric acid was added during analysis to avoid any reduction of Se(VI) (final concentration of 1.2 M HCl).
Vehicle:
no
Details on test solutions:
see test conditions
Test organisms (species):
Chlamydomonas reinhardtii
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP, Cumbria, United Kingdom, culture 11/32B)
- Age of inoculum (at test initiation): cultures in end of exponential growth phase
- Method of cultivation: Maintenance and experimental culturing were carried out axenically in a modified high salt medium under constant illumination at 100 µmol photons/m2/s, at an ambient temperature of 25±0.5 °C, and with rotary agitation (∼120 rpm).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
Can be calculated (0.002 M Ca and 0.00104 M Mg)
Test temperature:
25 °C
pH:
7
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
0, 10, 50, 100 and 500 µM (nominal)
0, 9.44 (±0.05), 48.6 (±0.5), 96.6 (±1.2) and 467 (±8) µM (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: 200-mL polycarbonate Erlenmeyer flask containing 150 mL of test solution
- Initial cells density: 2500 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: no, modified high salt medium adapted from Harris (1988)

TEST MEDIUM / WATER PARAMETERS
- Modified high salt medium composed using deionized water as dilution water
- Detailed composition given in Table 1
- Sulfate = 0.0812 mM

OTHER TEST CONDITIONS
- Adjustment of pH: yes, daily using 0.1 M KOH
- Light intensity and quality: 100 µmol photons/m²/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell abundance and average cell diameter were measured daily for each experimental culture using a Coulter Z2 particle counter (1:10 dilution with ISOTON II isotonic solution, using a 100 µm orifice tube; Beckman Coulter, Roissy, France).

TEST CONCENTRATIONS
- Test concentrations: 0, 10, 50, 100, 500 µM
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
790 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
other: Growth (maximal population density)
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
3 950 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
other: growth (maximal population density)
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
6 320 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
other: Growth (maximal population density)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 39 500 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Remarks:
Se
Basis for effect:
growth rate
Reported statistics and error estimates:
The Excel macro REGTOX, based on the Marquardt algorithm, was used to calculate ECx values. Confidence intervals were estimated by bootstrap non-parametric simulation. An analysis of variance completed by lowest significant difference post-hoc comparisons was performed to test for significant differences.
Conclusions:
Reliable (Klimisch 2) study on the toxicity of sodium selenite to Chlamydomonas reinhardtii. The 96-h NOEC, LOEC and EC50 were 790, 3950 and 6320 µg Se/L, respectively, and were based on growth (maximal population density). No significant effect was observed on the exponential growth rate up to the largest dose of 39500 µg Se/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: The growth of Chlamydomonas reinhardtii under different selenate and sulphate concentrations was determined
- Short description of test conditions:Experiments were carried out over a 96 h period on 200 mL of culture medium containing 8 or 80 mol L−1 of sulphate ions. he five selenate concentrations were 0 (untreated), 0.01, 0.10, 0.60, 2.5 mol L−1 in medium containing 8 mol L−1 sulphate ions and 0 (untreated), 0.60, 2.5, 6.0, 11.5 mol L−1 in medium with a final concentration of 80 mol L−1 sulphate ions.
- Parameters analysed / observed: Algal density was estimated each day by measuring absorbance at 690 nm.
GLP compliance:
not specified
Remarks:
This study was not performed for the purpose of fulfilling REACH requirements; results were identified in a scientific journal.
Specific details on test material used for the study:
Supplier: Sigma Aldrich, Steinheim, Ger- many
Analytical monitoring:
yes
Details on sampling:
The concentrations of selenium and selenate stability in the medium were measured at the beginning and at the end of each experiment. Total selenium in algae was measured at the end of the 96 h exposure period.

Vehicle:
no
Details on test solutions:
The range of selenate concentrations was obtained from an aqueous stock solution
Test organisms (species):
Chlamydomonas reinhardtii
Details on test organisms:
TEST ORGANISM
- Strain: 11/32B
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP, Cumbria, United King- dom).
- Method of cultivation: Cells were cultivated in sterile conditions in glass Erlenmeyer flasks (250 mL) equipped with stoppers and containing 200 mL of a modified high salt medium (Table 1). Cultures were grown in incubators at 25 ± 0.5 ◦ C, on a flat bed orbital shaker (120 rpm) with constant illumination (100 mol m−2 s−1 of photosyntheti- cally available radiation). To maintain exponential growth, cultures approaching end-growth phase were inoculated in a new culture medium to give 2500 cells mL−1 .
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
pH:
7
Nominal and measured concentrations:
0 (untreated), 0.01, 0.10, 0.60, 2.5 µmol L−1 in medium containing 8 µmol L−1 sulphate ions and 0 (untreated), 0.60, 2.5, 6.0, 11.5 µmol L−1 in medium with a final concentration of 80 µmol L−1 sulphate ions.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass Erlenmeyer flasks (250 mL)
- Type (delete if not applicable): closed
- Initial cells density: 2500 cells mL−1.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: no, modified high salt medium
- Detailed composition if non-standard medium was used:
Components Nominal concentrations (mol L−1 )
NH4 9.36 × 10−3
Cl 13.47 × 10−3
K 1.22 × 10−3
PO4 0.14 × 10−3
Cinorg 50 × 10−6 (pCO2 = 3.5 × 10−4 atm)
NO3 2.92 × 10−3
SO4 81.2 × 10−6 / 8.1 x 10-6 (high / low sulphate medium)
Mg 1.04 × 10−3
Ca 2.00 × 10−3
Naa 0.10 × 10−3
BO3 3.01 × 10−6
Mn 2.09 × 10−6
EDTA 8.06 × 10−7
Fe 5.92 × 10−7
MoO4 3.00 × 10−8
Zn 2.43 × 10−8
Co 1.09 × 10−8
Cu 7.04 × 10−11

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, to pH 7 with KOH (0.1 mol L−1 ) each day
- Photoperiod: 24h
- Light intensity and quality: 100 mol m−2 s−1 of photosynthetically available radiation

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer: measuring absorbance at 690 nm (Cecil 2020, Cecil Instruments Ltd, Cambridge, UK).

TEST CONCENTRATIONS
- Test concentrations: The five selenate concentrations were 0 (untreated), 0.01, 0.10, 0.60, 2.5 µmol L−1 in medium containing 8 µmol L−1 sulphate ions and 0 (untreated), 0.60, 2.5, 6.0, 11.5 µmol L−1 in medium with a final concentration of 80 µmol L−1 sulphate ions.
Key result
Duration:
96 h
Dose descriptor:
EC50
Remarks:
For normal sulphate concentrations (80 µmol/L)
Effect conc.:
244.8 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Se
Basis for effect:
other: Growth (maximal cell density)
Remarks on result:
other: confidence limits: 130.3 - 383.7 µg Se/L
Duration:
96 h
Dose descriptor:
EC50
Remarks:
for low sulphate conditions (8 µmol/l)
Effect conc.:
31.6 µg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Se
Basis for effect:
other: Growth (maximal cell density)
Remarks on result:
other: confidence limits: 19.0 - 41.1 µg Se/L
Reported statistics and error estimates:
The 50% Effect Concentrations and associated 95% confidence intervals estimated at 8 and 80 mol L−1 sulphate ions were 0.40 [0.24–0.52] µmol L−1
and 3.10 [1.65–4.86] µmol L−1, respecively.

The concentrations of total dissolved selenium in the culture medium were constant throughout the 96 h exposure period, the decrease being less than 5%. In parallel, to check for possible changes in selenium species distribution, several replicates of culture medium samples were analysed by coupled HPLC/ICP-MS. The results confirmed that ambient selenium remained in the sele- nate form throughout the whole 96h experimental period. No other forms (selenite, selenocystine nor selenomethionine) were detected.

Conclusions:
Reliable (Klimisch 2) study on the toxicity of selenate to the marine alga Chlamydomonas reinhardtii under different sulphate conditions. The 96-h EC50 for growth was 0.40 [0.24–0.52] µmol L−1 (31.6 µg Se/L) and of 3.10 [1.65–4.86] µmol L−1 (244.8 µg Se/L) of ambient selenate were obtained, at 8 and 80 µmol L−1 of sulphate ions in the medium, respectively. The value of 244.8 µg Se/L is selected for classification and risk assessment because of the representative sulphate concentration in the exposure medium.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
GLP compliance:
not specified
Remarks:
This study was not performed for the purpose of fulfilling REACH requirements; results were identified in a scientific journal.
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Microcystis aeruginosa
Details on test organisms:
TEST ORGANISM
- Strain: FACHB-942
- Source (laboratory, culture collection): Freshwater Algae Culture Collection at the Institute of Hydrobiol- ogy (Wuhan, China).
- Age of inoculum (at test initiation): the inoculum was taken from a culture in the log growth phase
- Method of cultivation: BG11 medium. Algae were cultured under the condition of 25 ± 2 C in 24-h photoperiod with cool white light (1500 lux) for 4 weeks (late exponential growth phase). Ampicillin (60 mg/L) was added into the culture medium during growth to control microbial contamination

ACCLIMATION
- Acclimation period: /
- Culturing media and conditions (same as test or not): same
Test type:
static
Water media type:
freshwater
Remarks:
BG11 medium
Limit test:
no
Total exposure duration:
96 h
Test temperature:
25 ± 2 C
Nominal and measured concentrations:
0 (control), 0.1, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0 and 10.0 mg Se/L, each replicated three times.
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Pyrex glass bottle
- Initial cells density: 0.5 * 10^6 cells/ mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes: BG11 medium


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24-h photoperiod
- Light intensity and quality: cool white light (1500 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: fluorimeter

TEST CONCENTRATIONS
- Test concentrations: 0 (control), 0.1, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0 and 10.0 mg Se/L,
Reference substance (positive control):
no
Duration:
4 d
Dose descriptor:
IC50
Effect conc.:
2.68 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element (total fraction)
Remarks:
Se
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% confidence interval: 1.66 - 3.55 mg Se /L
Reported statistics and error estimates:
95% confidence interval, 1.66 - 3.55 mg Se/L
Conclusions:
Reliable (Klimisch 2) study on the toxicity of selenite to the cyanobacteria Microcystis aeruginosae. The 96-h IC50 was determined to be 2.68 (1.66 - 3.55) mg Se/L.

Description of key information

Reliable NOEC and EC50 values for freshwater algae and cyanobacteria range from 197 to 4570 and from 245 to 44240 µg Se/L, respectively.

The most critical NOEC and EC50 were 197 and 245 µg Se/L, respectively, and represent effect concentrations for maximal cell density of the unicellular green alga Chlamydomonas reinhardtii from 96 -h studies with disodium selenate.

The NOEC and EC50 for saltwater algae were 11000 and 45000 µg Se/L, respectively, and represent growth-rate based effect concentrations from a 96-h study on the toxicity of selenate to the halophilic Dunaliella viridis.

All reliable chronic NOEC values were used for the derivation of a PNECwater based on the statistical extrapolation approach.

Key value for chemical safety assessment

EC50 for freshwater algae:
245 µg/L
EC50 for marine water algae:
45 000 µg/L
EC10 or NOEC for freshwater algae:
197 µg/L
EC10 or NOEC for marine water algae:
11 000 µg/L

Additional information

Reliable data are available for Na2SeO3 and Na2SeO4 and both for freshwater and saltwater algae. The freshwater algae represented are the unicellular green algae Selenastrum capricornutum and Chlamydomonas reinhardtii. The halophilic Dunaliella viridis is considered representative for saltwater algae. Additionally, some studies are identified reporting relevant information for cyanobacteria (Microcystis aeruginosa and Anabaena flos-aquae). One study reports 10-d NOEC values for Anabaena flos-aquae obtained from experiments with Na2SeO3, Na2SeO4 and seleno-L-methionine (Kiffney and Knight, 1990). The NOEC values from these experiments are based on chlorophyll a content and are reported to be 1030, 995 and 30.5 µg Se/L, respectively. The data for Se-(D)L-methionine are not taken into account for the assessment of direct effects of inorganic selenium compounds to aquatic organisms because there is some concern on different biochemical behaviour of this selenium containing amino acid compared to inorganic Se compounds. All results are based on added dissolved Se concentrations.

NOEC and EC50 values for freshwater algae range from 197 to 4570 and from 245 to 44240 µg Se/L, respectively. The lowest NOEC and EC50 are growth-based effect concentrations for maximal cell density of the unicellular green alga Chlamydomonas reinhardtii from 96-h studies with selenate (Geoffroy et al., 2007; Fournier et al., 2010). The highest NOEC and EC50 are reported for a standard OECD 201 conform 72-h growth-rate inhibition study with sodium selenite and the unicellular green alga Selenastrum capricornutum (LISEC, 1992). Brix et al. (2004) reported the only available reliable data for a saltwater species (the halophilic alga Dunaliella viridis). The growth rate-based 96-h NOEC and EC50 for Se as selenate were reported to be 11000 and 45000 µg Se/L, respectively. Selenium toxicity appears to be highly species and endpoint-specific. Based on the data available, no conclusions could be made on the relative toxicity of selenite and selenate.