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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-10-06 to 2009-10-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to current OECD-Guideline and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tetrachloro-p-benzoquinone
- Molecular formula: C6Cl4O2
- Molecular weight: 245.88 g/mol
- Physical state: Solid, yellow crystalline powder
-

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: The concentrations of the test item were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: At test start two samples of the saturated solution, before and after adjusting the pH value, were prepared and analysed. Separate samples without algae were prepared at test start. At end of the test samples were taken directly from the test replicates.
- Sample storage conditions before analysis:All samples were stored at 6 ± 2 °C until start of analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution* of 40 mg/L was prepared with dilution water. The pH value of the dilution water was adjusted to
pH 6 ± 0.2 (by addition of 1 N NaOH and HCl) because the substance is stable under these conditions. The dispersion was shaken with 20 rpm for 24 h at ambient temperature and filtrated with 0.45 µm RC filter. Prior to preparing the dilution levels the saturated solution was adjusted to pH 8.1 ± 0.2 by addition of 1 M NaOH.
- Eluate: Dilution water
- Differential loading: The saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor 2: 1 - 3.2 - 10 - 32 and 100 % of the saturated solution corresponding to initially measured concentrations of 0.0115 – 0.0364 – 0.115 – 0.373 – 1.23 mg/L.
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms

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 from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
Not measured
Test temperature:
Mean: 22 °C, Min: 21 °C, Max: 23 °C
pH:
Initially measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
1.23 7.98 8.08
0.373 8.12 8.35
0.115 8.08 8.48
0.03641) 7.92 8.57
0.01151) 8.01 8.46
Control 8.07 8.73
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to "Any other information on material and methods"
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- 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: 5074 cells/mL
- Control end cells density: Mean 1004482 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0-72 h)

Dilution level out of the saturated solution
[%] Rate Related Inhibition [%] Yield Inhibition[%]
100 9 39
10 3 15
1 1 7
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.23 mg/L
Nominal / measured:
meas. (initial)
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: Yes
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.600 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.280 (CI 0.266 - 0.299) mg/L
Reported statistics and error estimates:
NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was =0.05.
Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % were marked biological significant.

Any other information on results incl. tables

NOEC, LOEC and EC - values of Tetrachloro-p-benzoquinone(0-72 h)

                   based on initially measured test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

1.23

LOEC

> 1.23

ErC10

> 1.23

ErC20

> 1.23

ErC50

> 1.23

Inhibition of Yield

NOEC

0.373

LOEC

1.23

EyC10

0.373 EC10 1.23

EyC20

0.373 EC20 1.23

EyC50

> 1.23

Cell Densities

 

Initially measured test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

1.23

1

5074

22930

117931

607680

 

2

5074

20580

120382

598414

 

3

5074

20027

132592

629643

 

Mean

5074

21179

123635

611912

 

0.373

1

5074

28674

263669

1175526

 

2

5074

32524

244559

1128716

 

3

5074

31786

268319

1163951

 

Mean

5074

30995

258849

1156064

 

0.115

1

5074

36919

235281

1040267

 

2

5074

35078

271636

1238674

 

3

5074

33077

256490

1012813

 

Mean

5074

35025

254469

1097251

 

0.03641)

1

5074

31630

174666

970460

 

2

5074

38264

269024

1130606

 

3

5074

35693

247892

884364

 

Mean

5074

35196

230527

995143

 

0.01151)

1

5074

37083

212350

1113415

 

2

5074

31593

253436

1033444

 

3

5074

31043

212399

818813

 

Mean

5074

33240

226062

988557

 

Control

1

5074

40215

233768

1174037

 

2

5074

34188

193051

928681

 

3

5074

36587

209992

886258

 

4

5074

36115

241693

1130983

 

5

5074

36082

180509

965495

 

6

5074

32511

230763

941436

 

Mean

5074

35950

214963

1004482

 

Evaluation after 72 h

                   Statistically significant differences of growth rates and yield compared to

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

Initially measured test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

1.23

1

1.60

9.41

602606

39.7

2

1.59

9.70

593340

40.6

3

1.61

8.74

624569

37.5

Mean

(+)*

1.60

9.28

(+)

606838

39.3

0.373

1

1.82

-3.08

1170452

-17.1

2

1.80

-2.31

1123642

-12.4

3

1.81

-2.90

1158877

-16.0

Mean

(-)

1.81

-2.76

(-)

1150990

-15.2

0.115

1

1.77

-0.77

1035193

          -3.58

2

1.83

-4.07

1233600

-23.4

3

1.77

-0.26

1007739

          -0.83

Mean

(-)

1.79

-1.70

(-)

1092177

          -9.28

0.03641)

1

1.75

0.55

965386

           3.40

2

1.80

-2.34

1125532

-12.6

3

1.72

2.31

879290

12.0

Mean

(-)

1.76

0.17

(-)

990069

            0.93

0.01151)

1

1.80

-2.05

1108341

-10.9

2

1.77

-0.64

1028370

           -2.90

3

1.70

3.76

813739

18.6

Mean

(-)

1.76

0.36

(-)

983483

            1.59

Control

1

1.82

1168963

2

1.74

923607

3

1.72

881184

4

1.80

1125909

5

1.75

960421

6

1.74

936362

Mean

(-)

1.76

(-)

999408

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

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 h)

SD

±

CV
[%]

Mean CV [%]

section-by-section                           

0 - 24 h

24 - 48 h

48 - 72 h

Control

1

2.07

1.76

1.61

1.82

0.233

12.8

13.1

2

1.91

1.73

1.57

1.74

0.169

9.71

3

1.98

1.75

1.44

1.72

0.269

15.6

4

1.96

1.90

1.54

1.80

0.227

12.6

5

1.96

1.61

1.68

1.75

0.187

10.7

6

1.86

1.96

1.41

1.74

0.295

16.9

Mean

1.76

 

SD ±

0.04

 

CV [%]

2.17

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study Tetrachloro-p-benzoquinone was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 1.23 and 0.373 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were > 1.23 and 1.23 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were > 1.23 mg/L, respectively which represents the maximum saturation under test conditions.
All effect levels are given based on initially measured concentrations of Tetrachloro-p-benzoquinone.
Executive summary:

The toxicity of Tetrachloro-p-benzoquinone(batch number INCB 5070661) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 , from October 06 to 09, 2009. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h. The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 103cells/mL. Based on a preliminary test, a definitive test with the saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor of 2, nominal: 1 - 3.2 - 10 - 32 - 100 % of the saturated solution corresponding to initially measured test item concentrations of 0.0115 – 0.0364 – 0.115 – 0.373 – 1.23 mg/L. The two lowest concentrations were calculated based on the results of the third lowest test item concentration with the dilution factor of √10. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Tetrachloro-p-benzoquinonewere analysed at all concentration levels after 0 and 72 h via HPLC analysis. The two lowest dilution levels were below the LOQ of   0.075 mg/L at test start. The measured concentrations for the two highest concentrations of Tetrachloro-p-benzoquinoneat test end were in the range of 31 – 67 % of the initially measured values. Concentrations of the lower concentrations were less than the LOQ of 0.075 mg/L. All effect values are given based on initially measured concentrations of the test item.

Table 1:    NOEC, LOEC and EC - values of Tetrachloro-p-benzoquinone(0-72 h)

                   based on initially measured test item concentrations [mg/L]

                               

Rate-related Inhibition

NOEC

1.23

LOEC

> 1.23

ErC10

> 1.23

ErC20

> 1.23

ErC50

> 1.23

Inhibition of Yield

NOEC

0.373

LOEC

1.23

EyC10

0.373 EC10 1.23

EyC20

0.373 EC20 1.23

EyC50

> 1.23