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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:
2014-04-01 to 2014-03-31
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The water accommodated fraction (WAF) was prepared with a nominal loading of the test item of 100 mg/L.
- Sampling method: The concentrations of the test item were analytically determined from the limit loading, the pH-control and the control after 0 hours (test start) and 72 hours (test end) via photometric analysis. Samples at test start were taken from an additional replicate without algae. At the end of the test samples were taken from pooled replicate with algae.
- Sample storage: All original samples were stored in a refrigerator until sample preparation. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The water accommodated fraction (WAF) was prepared with a nominal loading of the test item of 100 mg/L.
100 mg/L of the test item were weighed out and transferred with an appropriate amount of dilution water into a brown glass flask. This dispersion was shaken for 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the aqueous phase or WAF was removed by siphoning (from the approximate centre of the glass flask) to give the 100 mg/L loading rate WAF.
- Eluate: Dilution water
- Differential loading: None, limit test
- Controls: Six replicates (without test item) were tested under the same test conditions as the test vessels.
- pH-Controls: Six replicates, neutralized to pH 8.1 ± 0.2 with NaOH/HCl
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
not specified
Test temperature:
min.: 22.0 max.: 23.0, mean value: 22.5 °C
pH:
Nominal test item loading pH-value
[mg/L] Start; 0 hours End; 72 hours
100 10.58 8.38
100, pH-control 8.13 8.48
Control 8.18 8.52
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
See window "Any other information on materials and methods incl. tables"
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 4998 cells/mL
- Control end cells density: Mean 920143 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per pH-control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes, 8.1 ± 0.2 for pH-control samples
- Photoperiod: 24 h
- Light intensity and quality: 4440 - 8880 lux, corresponding to 60 - 120 µE x m^-2 x s^-1, mean value: 5225 lux



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS

Results of the Range Finding Test (0 - 72 hours)
Nominal Test Item Loading
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 (pH adjusted) 0 1
100 2 12
10 4 20
1 6 27
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
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):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes

Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.684 0.684 ± 0.281
95 % confidence interval 0.657 - 0.714 0.684 ± 0.281
Yield Inhibition
EyC50 0.315 0.314 ± 0.087
95 % confidence interval 0.297 - 0.3355 0.314 ± 0.087

SD = Standard deviation
Reported statistics and error estimates:
NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEL/LOEl calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha=0.05.

Cell Densities

Nominal Test Item Loading

Replicate

Cell Density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

100

1

4998

26587

164666

719393

2

4998

21181

150745

639043

3

4998

22970

139301

641815

4

4998

22216

148224

637571

5

4998

20730

151057

664642

6

4998

22155

167170

665162

Mean

4998

22640

153527

661271

100 neutralized

(pH-control)

1

4998

37017

193159

884813

2

4998

30164

192154

774316

3

4998

35964

194865

727233

4

4998

31741

166321

756947

5

4998

34704

200713

729009

6

4998

30017

183075

681839

Mean

4998

33268

188381

759026

Control

1

4998

37229

182304

908159

2

4998

30654

199552

835217

3

4998

27877

221097

976857

4

4998

29211

200427

931636

5

4998

27812

191530

883470

6

4998

32638

205495

985520

Mean

4998

30904

200068

920143

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal Test Item Loading

Replicate

Growth Rate

Growth Rate Inhibition

Yield

Yield Inhibition

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

1.66

5

 

714395

22

2

 

1.62

7

 

634045

31

3

 

1.62

7

 

636817

30

4

 

1.62

7

 

632573

31

5

 

1.63

6

 

659644

28

6

 

1.63

6

 

660164

28

Mean

(+)

1.63

6

(+)

656273

28

100 neutralized (pH-control)

1

 

1.73

1

 

879815

4

2

 

1.68

3

 

769318

16

3

 

1.66

4

 

722235

21

4

 

1.67

4

 

751949

18

5

 

1.66

4

 

724011

21

6

 

1.64

6

 

676841

26

Mean

(+)

1.67

4

(+)

754028

18

Control

1

 

1.73

 

 

903161

 

2

 

1.71

 

 

830219

 

3

 

1.76

 

 

971859

 

4

 

1.74

 

 

926638

 

5

 

1.73

 

 

878472

 

6

 

1.76

 

 

980522

 

Mean

 

1.74

 

 

915145

 

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0 – 72 hours)

 

Replicate No.

Specific Growth Rate [d-1]

Mean

(0 - 72 h)

SD

±

CV
[%]

Mean CV [%]

Section-by-Section                           

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

2.01

1.59

1.61

1.73

0.237

13.7

12.8

2

1.81

1.87

1.43

1.71

0.240

14.0

3

1.72

2.07

1.49

1.76

0.295

16.8

4

1.77

1.93

1.54

1.74

0.196

11.2

5

1.72

1.93

1.53

1.73

0.201

11.6

6

1.88

1.84

1.57

1.76

0.169

 9.58

 

 

 

Mean

1.74

 

 

 

 

 

SD ±

0.02

 

 

 

 

CV [%]

1.20

 

Validity criteria fulfilled:
yes
Conclusions:
In this study celitement was found slightly to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for both, inhibition of growth rate and inhibition of yield, after 72 hours was < 100 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 100 mg/L, respectively.
All effect levels are given based on the nominal concentration of celitement.
Executive summary:

The toxicity of celitement to the unicellular freshwater green alga Desmodesmus subspicatuswas determined according to the principles of OECD 201 (2011) at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from2014-03-31 to 2014-04-04 with the definitive exposure phase from 2014-04-01 to 2014-04-04. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

 

The test item is a mixture, solid and insoluble in water. In view of the difficulties associated with the evaluation of aquatic toxicity of complex mixture test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose the organisms to a Water Accommodated Fraction (WAF*) of the test item in cases where the test item is a complex mixture. Exposures are expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

 

The study was conducted under static conditions with an initial cell density of 4998 cells/mL. One limit loading of 100 mg/L of the test item was prepared. The test item was applied onto a glass slide. The glass slide with the test item was inserted in a brown glass flask with an appropriate amount of dilution water. This dispersion was shaken for 24 hours with 20 rpm at room temperature.After a separation phase of 1 hour,the aqueous phase or WAF was removed by siphoning (from the approximate centre of the glass flask) to give the 100 mg/L loading rate WAF. The water soluble fraction was clear. Six replicates were tested for the limit loading, the pH - neutralized limit loading (pH – control) and for the control. Environmental conditions were within the acceptable limits.

 

The concentrations of the test item celitement were analytically evaluated via photometric analysis of silicon at the start of the exposure (0 h) and at the end of the exposure (72 h) of the limit loading level, the pH-control and the control.

The measured silicon concentration at the start of the exposure (0 h) was 7.45 mg/L in the limit loading level and 7.65 mg/L in the pH-control. At the end of the exposure the measured silicon concentrations gave recoveries of 98 and 102 % of the initially measured values of the limit loading and the pH-control indicating the stability of the test item in the WAF during the exposure phase.All effect values given are based on the nominal test item loading of celitement.

* Water accommodated fractions (WAF): The aqueous medium containing only the fraction of a multi-component test item that is dissolved and / or present as a stable dispersion/emulsion under test conditions in the test medium, acc. to OECD Guidance Document, No. 23.

Description of key information

Short-term toxicity testing with the freshwater green alga Desmodesmus subspicatus was carried out according to the OECD 201 guidance taking into account GLP and thus resulting in a Klimish 1 score. No acute toxicity was observed.

Key value for chemical safety assessment

Additional information

The NOEC-value of the test item for both, inhibition of growth rate and inhibition of yield, after 72 hours was < 100 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 100 mg/L, respectively.