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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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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
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
OECD, 1996
Principles of method if other than guideline:
Copper toxicity to P. subcapitata was determined in water collected from 10 areas located throughout Europe and in artificial medium (ISO medium). The natural surface waters were collected from different eco-regions in Europe and have a different combination of pH, hardness and DOC. As an artificial medium, an ISO-medium (ISO, 1989) and ISO-medium with adjusted levels of Ca, Mg or pH were used.
Three pH-levels were tested: 5.8, 6.7 and 8. pH was buffered using 750 mg/l of MOPS (3-[NMorpholino]propanesulfonic acid). MOPS was chosen as buffer since it is reported to be completely non-complexing for metals and it is recommended by US-EPA (1991) as it does not change the toxicity of effluents and sediment pore waters.
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
Dissolved copper concentrations were determined at the beginning of the tests.
Vehicle:
not specified
Details on test solutions:
The nutrients were added and the test media were spiked with copper added as CuSO4. Then the pH of media were adjusted to their original value by adding 0.1 N NaOH or 0.1 N HCl The spiked media were equilibrated for 48 h at 25°C before testing started.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): the starter culture was obtained from the Culture Collection for Algae and Protozoa (CCAP 278/4, CEH, Windermere, UK)
- Culture: the algae were grown in ISO medium at 20 (+/- 1) °C under continuous illumination at 5000 lux and continuous aeration
- Density: At the beginning of the tests, the density of algae was 1x10^4 cells/mL in each test flask
- Only algae in log-phase were used in the tests,
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
ISO medium: 25 mg/L CaCO3
Natural waters: between 7.9 and 238 mg/L CaCO3
Test temperature:
23+-1°C
pH:
ISO medium: 7.8
natural waters: between 5.52 and 8.3
Nominal and measured concentrations:
The study was conducted with1 control and 5 test concentrations. The test concentrations not reported. The difference between the lowest and highest test concentrations was 1 order of magnitude.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass Erlenmeyer flasks
- Material, size, headspace, fill volume: 100 mL test vessels filled with 50 mL of test medium
- Aeration: none
- Initial cells density: 1x10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: ISO medium (1989)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The study was conducted in an artificial ISO medium and natural surface waters collected from 10 eco-regions in Europe.
Toxicity of Cu to P. subcapitata was determined in ISO-medium (ISO, 1989) and ISO-medium with adjusted levels of Ca, Mg or pH. 72 h-EbC50s were determined at calcium concentrations of 0.25 (unaltered ISO-medium), 1.0 and 2.5 mM and at magnesium concentrations of 0.25, 1.0 and 2.0 mM. Calcium and magnesium were added as CaCl2.2H2O and MgCl2.6H2O, respectively. Three pH-levels were tested: 5.8, 6.7 and 8. pH was buffered using 750 mg/l of MOPS (3-[NMorpholino]propanesulfonic acid).
Surface water samples were taken and transferred to the laboratory in 30 L polyethylene vessels within 24 h and stored at 4°C in the dark. Tests with natural waters were performed a maximum of 1 week after sampling. The natural waters were filtered over 0.45 µm filter to remove particulate material and possible local algae and nutrients similar to those present in the ISO medium were added.
- Dissolved organic carbon: between 1.6 and 20.4 mg/L (for natural waters); <1 mg/L (for ISO medium)

OTHER TEST CONDITIONS
- Adjustment of pH: only in surface waters, the pH was adjusted to its original field pH by addition of HCl or KOH
- Light intensity and quality: continuous illumination (4000 lux) measured at the surface of the light table

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The cell density was measured after 24, 48 and 72 h.
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not reported. The difference between the highest and lowest concentrations was 1 order of magnitude.
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
32 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Basis for effect:
biomass
Remarks on result:
other: other: confidence limits: 23-42 µg/L; Somerain, pH 6.39, hardness 27.8 mg/L CaCO3, DOC 1.55 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
152 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Basis for effect:
biomass
Remarks on result:
other: confidence limits: 108-216 µg/L. Bihain water: hardness 10.1 mg/L CaCO3, pH 5.94, DOC 8.91 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
194 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: confidence limits: 106-322 µg/L; Lake Skarsjon, pH 5.52, hardness 7.9 mg/L CaCO3, DOC 10.3 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
52.7 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: confidence limits: 48.8-57.5 µg/L; Lake monate; pH 8.23, hardness 48.5 mg/L CaCO3, DOC 2.52 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
65.4 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: confidence limits: 49-58 µg/L; Maarkermeer; pH 8.3, hardness 238 mg/L CaCO3, DOC 8.24 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
33.9 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: confidence limits: 30-44 µg/L; River Rhine; pH 8.06, hardness 191 mg/L CaCO3, DOC 1.98 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
113 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: confidence limits: 63-167 µg/L; River Mole; pH 7.55, hardness 132 mg/L CaCO3, DOC 6.13 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
163 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: confidence limits: 117-213 µg/L; Ankeveense Plassen; pH 7.5, hardness 134 mg/L CaCO3, DOC 20.4 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
36.9 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: confidence limits: (34.4-39.8 µg/L; Lake Segrino; pH 8.2, hardness 169 mg/L CaCO3, DOC 1.7 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
245 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: confidence limits: 180-304 µg/L; Ankeveense Plassen; pH 7.3, hardness 166 mg/L CaCO3, DOC 17.8 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
97.4 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: confidence limits: not determined; Clywydog; pH 6.31, hardness 10 mg/L CaCO3, DOC 2.72 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
108 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: confidence limits: not determined; Maarkermeer; pH 8.26, hardness 220 mg/L CaCO3, DOC 6.42 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16.5 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: confidence limits: 11.7-21.3 µg/L; ISO medium, pH 7.8, DOC < 1 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.2 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: Somerain, pH 6.39, hardness 27.8 mg/L CaCO3, DOC 1.55 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
13.5 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Remarks on result:
other: ISO medium, pH 7.8, DOC < 1 mg/L
Reported statistics and error estimates:
The growth inhibition percentages compared to the control were calculated according to the ‘area under the curve’ method prescribed by the OECD (1996). The inhibition percentages were plotted against the measured dissolved copper concentrations and a sigmoidal concentration–response relationship was fitted through the data points using the software package Sigmaplot 4.0 (SPSS Inc., Chicago, IL, USA). From this relationship the 72 h-EbC50 and the 95% confidence intervals were derived. The NOEbC for each test medium was determined using ANOVA (p < 0.05).
Validity criteria fulfilled:
yes
Conclusions:
The 72-h EC50 values (72h) for P. subcapitata in the natural waters ranged between 32 µg/L and 245 µg/L and NOEC values varied between 4.2 µg/L and 164 µg/L. The 72-h EC50 in ISO medium was 16.5 µg/L and the NOEC was 13.5 µg/L.
Executive summary:

An acute toxicity study with Pseudokirchneriella subcapitata was conducted in accordance with OECD Guideline 201. The aim of this study was to investigate the individual effects of Ca, Mg (the hardness cations) and pH on the toxicity of copper in reconstituted artificial test waters and to study the copper toxicity in 13 spiked surface waters originating from different European eco-regions. Surface waters were selected such that a broad range of DOC (1.55–20.4 mg/l), pH (5.52–8.30) and water hardness (7–238 mg CaCO3/l) were covered.

The 72h-EC50 values for P. subcapitata in the natural waters ranged between 32 µg Cu/L and 245 µg/L and the NOEC values ranged between 4.2 µg Cu/L and 164 µg Cu/L at natural waters (pH 6.39, hardness 27.8 mg/L CaCO3, DOC 1.55 mg/L and at pH 7.3, hardness 166 mg/L CaCO3, DOC 17.8 mg/L, respectively).

The 72 -h EC50 in ISO medium was 16.5 µg Cu/L and NOEC was 13.5 µg Cu/L.

This is good quality experimental study conducted according to a standard method (OECD 201) and published in a peer-reviewed journal. The study is considered reliable with restrictions as there are minor restrictions in reporting (GLP status not known, test concentrations are not reported). 

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: Test conducted under GLP according to standard OECD guidelines
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control:
- frequency: at t=0, t=24h and t=72h
- volume: 4.8 mL
- storage: samples were stored in a freezer until analysis
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling
Details on test solutions:
Pre-culture medium: M2; according to OECD 201 Guideline, formulated using MilliQ tap water and subsequently passed over activated carbon and ion-exchange cartridges
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
In-house laboratory culture; strain NIVA CHL 1
Stock culture: Algae stock cultures were started by inoculating growrth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24 degrees Celcius.
Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 10,000 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Te cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
test medium (m2): 24 mg/L as CaCO3
pH:
test medium (M2): 8.1 +/- 0.2
Nominal and measured concentrations:
nominal: 0.1, 1, 10 and 100 mg/L of strontium nitrate
Measured 100 mg/L: 102.5 mg/L strontium nitrate (t=0)
Measured 100 mg/L: 104 mg/L strontium nitrate (t=24h)
Measured 100 mg/L: 107.5 mg/L strontium nitrate (t=72h)
Measured 100 mg/L (mean): 104.7 mg/L strontium nitrate, i.e., 43.3 mg Sr/L
Details on test conditions:
Vigorous shaking was sufficient to completely dissolve the test substance in medium
Test vessels: 100 mL; all glass - vessels were distributed at random in the incubator
Test volume per replicate: 50 mL
Control: test medium without test substance or other additives
No of replicates for control and highest test concentration: 6
No. of replicates for remaining test concentrations: 3
1 or 2 replicates of each test concentration without algae
1 replicate of each concentration for sampling purposes
Cell concentration at start: 10,000 cells/mL
Illumination: continuously using TLD-lamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range off 66 to 77 uE/m2/s
Algal cells were kept in suspension by continuous shaking

- pH was measured at the beginnig and end of the test period, and was not supposed to deviate by more tan 1.5 units during the test
- at the end of the final test microscopic observations were performed on the limit and one lower concenration to verify a normal and healthy appearance of the inoculum algae and to observe for any abnormal appearance of algae.
- Cells were counted using a microscope and a counting chamber at the beginning of the test; thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with a cuvette (pathlength : 10 mm). Quantification of cell density was based on a calibration curve.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 43.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 104.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 43.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 104.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 43.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 104.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 43.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 104.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
No significant differences were recorded between the values for growth rate or yield at the limit concentration, i.e., 100 mg/L when compared to the control group. The growth and yield in lower test concentrations were stimulated rather than reduced/inhibited by the test substance. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
Compliance with the Quality criteria regarding mainenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentrartion but without algae, and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Test results were in compliance with all validity criteria (minimum increase in cell density, criteria on variation (growt rates, section-by-section specific growth rates) were not exceeded.
Results with reference substance (positive control):
The 72h-EC50 for growth reduction was 1.2 mg/L (95%CL: 0.77-2.0 mg/L).
The historical range of this value lie between 0.82 and 2.3 mg/L.
Reported statistics and error estimates:
Recorded effects were not significant (< 10%); EC50 values could not be calculated because the test substance proved to be non-toxic within the tested concentration range.
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 are used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or yield inhibition (ANOVA, Tukey test, Bonferroni t-test, TOXSTAT Release 3.5, 1996, D.D. Gulley et al).

Percentage reduction of growth rate (total test period)

 Nominal Concentration Strontium nitrate (mg/L Mean growth rate - u (0 -72h)  Mean growth rate - reduction (%) 
Control  0.05924   
0.10  0.06675  - 12.7 
1.0  0.06704  -13.2 
10  0.06628  -11.9 
100  0.05931  -0.1 
     
Validity criteria fulfilled:
yes
Conclusions:
No statistically significant differences were found between the control and any test item concentration up to and including 100 mg test item/L. The results of the study demonstrate that the EC50 for Pseudokirchneriella subcapitata is >100 mg test item/L. The no-observed effect concentration (NOEC) is ≥104.5 mg/L strontium nitrate (based on measured concentrations). Considering the measured concentrations, the EC50 for Pseudokirchneriella subcapitata is > 43.3 mg Sr/L (measured in the highest concentration at test end). The no-observed effect concentration (NOEC) is ≥ 43.3 mg/L Sr.
These data can be used for hazard and risk assessment purposes (classification; PNEC-derivation)

Description of key information

For copper, the EC50 value of 32 µg Cu/L and NOEC of 4.2 µg Cu/L were determined in test medium at pH 6.39.

For strontium, the EC50 is >43.3 mg Sr/L and NOEC is ≥43.3 mg Sr/L.

Key value for chemical safety assessment

Additional information

There are no data available on acute toxicity to aquatic algae for strontium apatite copper doped.

Based on the transformation/dissolution (TD) tests conducted with strontium apatite copper doped (Martinez, 2016b), strontium apatite copper doped is expected to release the copper and strontium in the environment to a higher extent at pH 6 than at pH 8. Therefore, the results at pH 6 are taken into consideration as a worst-case scenario.

For copper and copper compounds, a large number of high quality published studies on toxicity to aquatic algae are available. The EC50 value of 32 µg Cu/L and NOEC of 4.2 µg Cu/L were determined at pH 6.39 in a study with Pseudokirchneriella subcapitata (Heijerick et al. 2005).

 

For strontium, the 72h-EC50 of > 43.3 mg Sr/L and NOEC of 43.3 mg Sr/L were determined in a guideline study with Pseudokirchneriella subcapitata (Tobor-Kaplon 2010).