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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 June 2016 - 22 August 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
yes
Remarks:
1) CV for mean specific growth rate for day 0-1 is not taken into account. 2) The test vessels were polystyrene disposable vessels instead of glass vessels.
Qualifier:
according to
Guideline:
other: OECD 23 (OECD series on testing and assessment guidance document on aquatic toxicity testing of difficult substances and mixtures.)
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 29: OECD series on testing and assessment no. 29: guidance document on transformation/disolution of metals and metal compounds in aqueous media.
Version / remarks:
2006
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Solubility in water: insoluble
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: WAFs were prepared by adding the test substance to 500 mL of OECD medium (OECD 201), subsequently the suspensions were stirred for 7 days at room conditions. Afterwards the suspension were filtered (0.454 µM) and the eluates were used in the tests.
- Controls: OECD medium (batch 5/07/2016) was used as control solution
- Evidence of undissolved material: no, eluates were clear and colourless
- Other: On days 2 and 3 some stirrers had stopped overnight and had to be reactivated again. Besides that, stirring was continuously.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Algae
- Strain: CODE CCAP: 278/4
- Source: VITO laboratory
- Age of inoculum (at test initiation): not indicated
- Method of cultivation: not indicated

ACCLIMATION: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23°C +/- 2°C
pH:
Between 7.79 and 8.12 at the start of the test.
Nominal and measured concentrations:
Nominal concentrations: 0, 63.4, 125.6, 250, 500.4, 1008.2 mg/L
Measured concentrations: not applicable, results were based on nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL polystyrene disposable vessels, open, fill volume: 100 mL medium or test dilution and 100 µL algal inoculum of a 13.3 x 10^6cells/mL stock
- Aeration: not reported
- Initial number of cells as reported: 229
- Control end number of cells as reported: 4203
Reported cell numbers presumably represent number of cells x 10^4 per test vessel (100 mL). This is not specified in the report.
- No. of cells per vessel: 13.3*10^3 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli RO, ~20µS
- Culture medium different from test medium: not indicated

OTHER TEST CONDITIONS
- Sterile test conditions: not indicated
- Adjustment of pH: no
- Light intensity and quality: continuous light, 3338 lux (at start)
- Other: continuous shaking (100 rpm)

EFFECT PARAMETERS MEASURED: biomass at 24, 48 and 72 h.
- Other measurements: pH was measured at the beginning and at the end of the test period (only measurements in the beginnng were reported)
- Determination of cell concentrations: at day 1, the number of cells in the pre-culture is measured with a coulter counter (BIO1518). Further measurements are surrogate measures using fluorescent signal for biomass indications: 800 µL samples of each infividual test vessel is transferred to the individual wells of a 24 well plates. Fluorescent signal in each well is measured by fluoroskan plate reader (excitation 444 nm, emission 680 nm, integration in 20 mSec). On day 1 a calibration curve is established between coulter counter and number of cells and fluorescent signal. This resulted in a correlation factor of 635.77. All fluorescence values that are generated in this test were multiplied by 635.77 to provide an estmation of the according cell numbers (biomass).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: no range find study was conducted
Reference substance (positive control):
yes
Remarks:
ZnCl2 (February 2016)
Key result
Duration:
72 h
Dose descriptor:
EC50
Remarks:
As reported
Effect conc.:
842 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Remarks:
As concluded
Effect conc.:
< 842 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: EC50 could not be determined because exact exposure values were not determined in this study.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
63.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities: no
- Other: final result (EC50) was determined by linear interpolation with the results from nominal concentrations.
- Effect concentrations exceeding solubility of substance in test medium: yes, solubility of test item is unknown, but test solutions were prepared in such a way that the soluble fraction remained. However, effect concentrations are nominal values and thus exceeding solubility.
- Reported EC50 deviates from conluded EC50 because no analyses was conducted on the test solutions so it was not proven that the algae were exposed to the reported 842 mg/L.
- No effect was reported below a loading rate of 125.6 mg/L. The NOEC was concluded to be 63.4 mg/L because at this loading rate no effect was statistically proven (p > 0.05).
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- 72h-EC50: 0.078 mg/L
- Result is within the historical range (0.06-0.168: mean value 0.107 mg/L; n=10) and just below the values provided by the ringtest organization Aquacheck (0.082-0.133: mean value 0.108 mg/L; n=8).
Reported statistics and error estimates:
- An excel template was used for the calcultations.
- The offset value (intercept) of the correlation curve between coulter counter number of cells and fluorescent signal, was not used to determine cell numbers. The report says it had no influence on the relative differences in cell numbers between test conditions and control because the intercept is seen as a constant value.
- Growth rate is calculated according to the method described in OECD guideline 201 (2006), using the biomass equivalent data.
- Student t-test was used to evaluate statistic relevant differences when compared to the controls.

For more detailed results, refer to the attached document (ALGE16022 - Tables.docx).

Validity criteria fulfilled:
yes
Remarks:
1) Biomass of the control increased more than 16x. 2) CV for the mean specific growth rate for individual days < 35%. 3) CV for specific growth rate for day 0-3 for replicates < 7%.
Conclusions:
The EC50 was reported as 842.0 mg/L and concluded was that the test substance Bismuth oxy-iodide bromide contains leachable compounds with inhibiting effects on algal growth. However, no analysis was conducted to determine actual concentrations in the test solutions. Also, the mean % inhibition of the specific growth rate for the test solution containing 250 mg/L was not taken into account for the conclusion. Therefore, the reported EC50 of 842.0 mg/L should be considered to be an estimation. This study shows that at a loading rate of 63.4 mg/L, no adverse effects were seen on the growth rate of algae (p > 0.05). The inhibition of specific growth rate was 3.6% at a loading rate of 125.6 mg/L, higher loading rates showed inhibition between 40.1% and 67.9% but overall not clear dose-respons relation could be seen. Therefore, no definite EC50 can be concluded.
Executive summary:

The aim of this test was to evaluate the effect of the water accommodated fraction (WAF) of the test substance (Bismuth oxy-iodide bromide) on the growth rate of exposed green unicellular algae Pseudokirchneriella subcapitata. This acute toxicity test was based on the OECD guidelines for testing of chemicals, no. 201 (2006). Test solutions were prepared by adding the test item to OECD M4 medium, followed by 7 days of stirring. After stirring the test substance was allowed to separate and the water fraction was collected. The water accommodated fraction was filtered (0.454 µm) prior to use. WAFs were prepared using 1008.2, 500.4, 250.0, 125.6 and 63.4 mg/L loading concentrations. No analysis was performed and results were based on nominal concentrations. Deviations on OECD 201 were considered acceptable and without effect on the final conclusion.

Growth rate after 72 hours was determined by daily measurements of biomass. Results showed there was an inhibiting effect at the two highest loading rates (1008.2 and 500.4 mg/L). The test solution containing 250.0 mg/L test substance, showed an inhibiting effect as well. However, this observation was not considered in the determination of the EC50 because the value was seen as aberrant as the percentage inhibition was higher compared to the percentages inhibition for higher loading rates. The EC50 was reported to be between 500.4 and 1008.2 mg/L loading rate, resulting in an EC50 value of 842 mg/L based on linear interpolation. However, because no analysis was conducted to determine actual test concentrations and the results were based on nominal values of a test substance with low solubility, the reported conclusion should be considered to be an estimation.

Statistically, no effect was reported at 63.4 mg/L (p > 0.05) mg/L. Inhibition of specific growth rate was 3.6% at a loading rate of 125.6 mg/L, higher loading rates showed inhibition between 40.1% and 67.9% but overall no clear dose-respons relation could be seen. Therefore, no definite EC50 can be concluded.

Description of key information

The aim of this test was to evaluate the effect of the water accommodated fraction (WAF) of the test substance (Bismuth oxy-iodide bromide) on the growth rate of exposed green unicellular algae Pseudokirchneriella subcapitata. This acute toxicity test was based on the OECD guidelines for testing of chemicals, no. 201 (2006). Test solutions were prepared by adding the test item to OECD M4 medium, followed by 7 days of stirring. After stirring the test substance was allowed to separate and the water fraction was collected. The water accommodated fraction was filtered (0.454µm) prior to use. WAFs were prepared using 1008.2, 500.4, 250.0, 125.6 and 63.4 mg/L loading concentrations. No analysis was performed and results were based on nominal concentrations. Deviations on OECD 201 were considered acceptable and without effect on the final conclusion.

Growth rate after 72 hours was determined by daily measurements of biomass. Results showed there was an inhibiting effect at the two highest loading rates (1008.2 and 500.4 mg/L). The test solution containing 250.0 mg/L test substance, showed an inhibiting effect as well. However, this observation was not considered in the determination of the EC50 because the value was seen as aberrant as the percentage inhibition was higher compared to the percentages inhibition for higher loading rates. The EC50 was reported to be between 500.4 and 1008.2 mg/L loading rate, resulting in an EC50 value of 842 mg/L based on linear interpolation. However, because no analysis was conducted to determine actual test concentrations and the results were based on nominal values of a test substance with low solubility, the reported conclusion should be considered to be an estimation.

Statistically, no effect was reported at 63.4 mg/L (p > 0.05) mg/L. Inhibition of specific growth rate was 3.6% at a loading rate of 125.6 mg/L, higher loading rates showed inhibition between 40.1% and 67.9%, but overall no clear dose-respons relation could be seen. Therefore, no definite EC50 can be concluded.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
63.4 mg/L

Additional information