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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:
2016-05-18 to 2016-09-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted: 23 March 2006
Annex 5 corrected: 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: H.C. Starck, 7160103
- Expiration date of the lot/batch: 18.04.2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store container tightly closed in dry place at room temperature
- Stability under test conditions: stable under storage conditions
- Solubility and stability of the test substance in the solvent/vehicle: Slightly soluble in water
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples were taken in triplicate from test dilution and the control at 0 h, 24 h, 48 h and 72 h from separate test vessels without algae. Additional samples at 72 h from test vessels with algae were filtered (Whatman CA-S, 0.45 μm).
In the test solution with a nominal loading rate of 100 mg/L no detection of zirconium and ytterbium could be measured.
- Sample storage conditions before analysis: Two replicates were kept in the refrigerator at 5 ± 3 °C until being sent to the analytical laboratory. The third sample was kept in the freezer at < -18 °C until finalization of the study.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A water accommodated fraction (WAF) with a loading rate of 111 mg/L was prepared with algal medium according to OECD 201. The WAF loading rate was chosen 1.11-fold higher than the nominal loading rate intended in the test solution to consider the dilution by the algal inoculum suspension. The 111 mg/L WAF was prepared by adding 111.2 mg of the test item to 1000 mL of algal medium. The test item was weighed of an inert PE-foil and then transferred together into the algal medium. Another vessel with 1000 mL algal medium and an inert PE-foil but without test substance was treated the same way (negative control). The WAF was shaken for 24 h in the dark at 21.6 – 22.1 °C. For shaking an overhead shaker was used (about 21 rpm). After the shake time undissolved material was allowed to sediment or float for a period of about 60 minutes. After this sedimentation period the WAF was clear. The test solutions were withdrawn from the middle of the suspension in the beakers using a glass tube and transferred into the test vessels.
- Differential loading: (nominal concentration): 0.0, 111 mg/L
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): After the shake time undissolved material was allowed to sediment or float for a period of about 60 minutes. After this sedimentation period, the WAF was clear.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Culture Collection of Algae at the University of Goettingen
- Method of cultivation: The strain used for this study has been cultured in the laboratory of Hydrotox GmbH since November 2015. Twice a week, the stock suspension is diluted into fresh Holm-Hansen medium under axenic conditions to keep it in exponential growth.

ACCLIMATION
- Acclimation period: Four days before starting the test, 5.6 mL of the algae stock suspension was diluted into 44.4 mL algal growth medium (according to OECD 201) to obtain a starting concentration of 1 x 10^5 algal cells per mL. They were incubated at 23.2 – 23.3 °C and 74.7 μE/m² s +/- 4.8 % (PAR). After 4 days, the cell concentration was determined using the Coulter Counter Z2. The cell concentration was 150.0 x 10^5 algal cells/mL. 0.700 mL pre-culture was added to 150 mL algal medium to obtain a nominal inoculum concentration of 0.7 x 10^5 algal cells/mL.
- Culturing media and conditions (same as test or not): algal growth medium according to OECD 201
- Any deformed or abnormal cells observed: not reported
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
23.1 – 23.4 °C
pH:
control: 7.96 at the beginning, 7.74 at the end
100 mg/L: 7.97 at the beginning, 7.72 at the end
Nominal and measured concentrations:
nominal: 100 mg/L
measured: measurements of all samples of zrconium and ytterbium were below the limit of quantification (< 1 µg/L). No detection of dissolved zirconium and ytterbium could be measured in 100 mg/L of the test samples.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks, wide necked
- Type (delete if not applicable): were sealed with a sterile cellulose stopper
- Material, size, headspace, fill volume: 100 mL
- Aeration: no
- Initial cells density: 0.7 x 10^5 cells/mL
- Control end cells density: 5.77 x 10^5
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: permanent
- Light intensity and quality: Light intensity was set on 45 % of the maximum light intensity resulting in a mean light intensity of 74.7 μE/m² s +/- 4.8 % (PAR)

CONDITIONS OF EXPOSURE
The test and control vessels (100 mL Erlenmeyer flasks, wide necked) were sealed with a sterile cellulose stopper and incubated in a light incubator RUMED 1301. This incubator provides stable temperature and light conditions. The test vessels are placed on a rotary disc made of acrylic glass, which provides space for 21 vessels on each of the four floors. The rotation of about 2 revolutions per minute (rpm) ensures that each vessel is exposed to equal light conditions. Each rotation includes 5 sudden stops by which the test suspensions are shaken. The flasks are illuminated laterally by eight fluorescent tubes (58 Watt each), which are separated from the incubation chamber by heat absorbing glass. Light intensity was set on 45 % of the maximum light intensity resulting in a mean light intensity of 74.7 µE/m^2 s +/- 4.8 % (PAR) (controlled by light intensity measurements with a spherical sensor on each of the four floors). The min/max temperature in a reference flask was recorded daily. During the time of exposure, the temperature was in the range of 23.1 to 23.4 °C. At the start of the test (0 h) and after 24, 48, and 72 h the biomass was determined by measuring the chlorophyll fluorescence for each test vessel. At the end of the test, the pH was measured in one flask from each nominal concentration.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: The fluorescence was measured with the microplate reader TECAN infinite F200 at an excitation wave length of 465 nm and an emission wave length of 670 nm. The cell concentration for the correlation factor is counted by Coulter Counter Z2.
- Chlorophyll measurement: At the start of the test (0h) and after 24, 48, and 72h the biomass was determined by measuring the chlorophyll fluorescence for each test vessel. For measuring the chlorophyll fluorescence of the algae in each test vessel 200 μL test medium was transferred into a 96-well micro-plate. The fluorescence was measured with the microplate reader TECAN infinite F200 at an excitation wave length of 465 nm and an emission wave length of 670 nm. Each measurement was conducted in duplicate. If the variation coefficient was > 10 % the measurement was repeated. Algal growth medium was measured as blank. The measured values were corrected for the blank values obtained with the algal medium. The dimensionless values for the fluorescence are a measure for the biomass (OECD 201). The measured chlorophyll fluorescence is converted into cell counts per mL by means of a correlation factor. This correlation factor is determined twice a year during quality check by measuring different cell densities with micro-plate fluorescence reader and coulter counter and subsequent calculation of a calibration curve. The algal count was determined for a cell size between 3 and 9 µm using a Coulter Counter Z2.
- Other: Correlation of chlorophyll fluorescence and cell count: To determine the conversion factor between the measured surrogate parameter chlorophyll fluorescence and the biomass, as required according to the OECD guideline, a correlation between fluorescence and cell count was provided. For this purpose, six dilutions with the nominal cell counts of 0.05 x 105, 0.1 x 105, 0.5 x 105, 1 x 105, 5 x 105 and 10 x 105 were prepared by diluting the pre-culture used for the test with algal medium. For all dilutions the cell count was determined with the Coulter Counter Z2, and the chlorophyll fluorescence was determined in the same way as in the test. Both values were correlated and the equation describing the curve was calculated. The correlation allows calculation the cell count from the measured surrogate parameter chlorophyll fluorescence.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test. No spacing factor
- Range finding study: No
Reference substance (positive control):
yes
Remarks:
potassium dichromate (Sigma, Steinheim, Germany, Lot No. MKBF2111V, from 12 January 2011)
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: ELR50
Effect conc.:
> 100 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 (for algal test): no abnormalities
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50 (Growth): 0.648 mg/L (95%-CI: 0.628 – 0.669 mg/L)
- EyC50 (Yield): 0.280 mg/L (95%-CI: 0.266 – 0.295mg/L)
- other: potassium dichromate (Sigma, Steinheim, Germany, Lot No. MKBF2111V, from 12 January 2011)
Reported statistics and error estimates:
The mean growth rate was similar in the test treatment compared to the control treatment (Table 1). The differences between control and test treatment were statistically not significant as was shown by student t-test analysis (Table 2).

Table 1: Mean growth rate and percent inhibition compared to the control

 

Test

Control

Nominal loading rate

100

0

Number of replicates

6

6

Mean growth rate

1.52

1.49

Mean inhibition of growth rate

- 1.97 %

 

 

Table 2: Result Student t-Test

Treatment [mg/L]

Mean

s2

df

% MDD

t

p(t)

Sign.

p(F)

Control

1.490

0.001

 

 

 

 

 

 

100.00

1.52

0.001

10

-2.329

1.57

0.926

-

0.32

-: non-significant

There is no statistically significant difference between Control and 100.00 mg/L.

Pair-wise comparison of treatments with "Control" by the t-test procedure. Significance was Alpha = 0.05, one-sided smaller; Mean: arithmetic mean of growth rates; n: sample size; s²: variance; % MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for Ho: µ1 = µ2; the differences are significant in case p(i) <= Alpha; p(F): probability of F computed by a F-test (Ho: var1 = var2 (homogeneity); p(F) > 0.05 is the criterion of variance homogeneity.

(The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).

Results of accompanying chemical analysis

The results of the elemental analysis of zirconium and ytterbium demonstrate that the solubility of both is below the limit of quantification (< 1 µg/L) in the test medium. In the test solution with a nominal loading rate of 100 mg/L no detection of zirconium and ytterbium could be measured.  Measurements of all samples of zirconium and ytterbium were below the limit of quantification (< 1 µg/L). No detection of dissolved Zirconium and Ytterbium could be measured in 100 mg/L of the test samples. The reference standard sample of Ytterbium of 1 mg/L was detected with 0.830 mg/L and within the accepted range of +/- 20 %. The reference standard samples of zirconium of 1 mg/L was detect with 0.720 mg/L. For a second measurement the reference standard and the samples of 0h of the control and 100 mg/L were measured. The standard was detected with 0.880 mg/L and was within the accepted range. The sample measurement was again below the LOQ.

Figure 1 (see below) shows the mean cell concentration of the test culture and the controls plotted against time of exposure in a logarithmic scale (growth curves). Algal growth is exponentially in both control and test treatment. The growth in the test treatment is similar compared to the control. In Appendix II, the determination of the conversion factor between cell count and measured chlorophyll fluorescence is presented.

 

Criteria of validity for this study (after 72 h of exposure)

- The biomass in the controls increased within 72 h from 0.07 x 105 algal cells/mL to 5.77 x 105 (factor 88) which is within the range for validity for the test (required ≥ 16).

- The maximum mean variation coefficient of the growth rate from day to day (day 0-1, 1-2 and 2-3) was 31.5 % in the controls which is within the validity range of 0 – 35 %.

- The variation coefficient calculated for the mean growth rate from day 0 to day 3 was 1.7 % and below the upper limit of 7 %.

--> The test is valid according to OECD 201.

Validity criteria fulfilled:
yes
Conclusions:
For the static algae growth inhibition test with Raphidocelis subcapitata, applying nominal concentrations of 0.0 (control), and 100 mg/L, the 72-h NOErLR is ≥ 100 mg/L (nominal loading).
Executive summary:

In a 72 hour acute toxicity study (algae growth inhibition test), the cultures of Raphidocelis subcapitata (Strain No. 61.81 SAG) were exposed to the test item at nominal concentrations of 0.0 (control) and 100 mg/L under static conditions in accordance with the OECD 201 (2006). The NOErLR value was ≥ 100 mg/L, the ErLR50 was > 100 mg/L based on cell density respectively. The nominal effective loading rate resulted in no inhibition of the growth rate.

There were no compound related phytotoxic effects.  

Accompanying chemical analysis revealed that the solubility of elemental zirconium and ytterbium released from the test item is below the limit of quantification (< 1 μg/L).

This toxicity study is classified as acceptable and satisfies the guideline requirements for an algae growth inhibition toxicity study.  

 

Results Synopsis

Test Organism: Raphidocelis subcapitata (Strain No. 61.81 SAG)

Test type: Static

- 72 h NOErLR: ≥ 100 mg/L (nominal loading)

- 72 h ErLR50 : > 100 mg/L (nominal loading)

Endpoint Effected: Growth rate

Description of key information

The 72-h NOErLR to cultures of Raphidocelis subcapitata determined according OECD guideline 201 is ≥ 100 mg/L (nominal).

Accompanying chemical analysis revealed that the solubility of elemental zirconium and ytterbium released from the test item is below the limit of quantification (< 1 μg/L).

Key value for chemical safety assessment

Additional information