Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-07-14 - 2008-07-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sample storage conditions before analysis: Room temperature, protected from moisture and light
Vehicle:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen

- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE•m-2•s-1 for 24 h per day.
- Culturing media: Nutrient medium Z according to LÜTTGE et al. (1994) :
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
4 - 12 d
Hardness:
0.24 mmol Ca+Mg/L
Test temperature:
Nominal range: 21 - 24 +/- 2 °C
Nominal and measured concentrations:
nominal: 11.5, 28.8, 72.0, 180 450 mg/l
The concentrations of the active ingredient Dimethylaminopropyl methacrylamide were analysed at all concentration levels at test start and test end via LC-MS. The measured concentrations at test start were in the range of 92 - 118 % of the nominal values. At the end of the test the test item gave recoveries of 89 – 101 %. All effect values are given based on nominal test item concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs
- Initial cells density: A three day old preculture was used as inoculum. Incubation was performed in 500 mL Erlenmeyer flasks with dilution water. For the start of the test the preculture was directly pipetted to the test and control replicates to receive an initial cell concentration of approximately 2 – 5 x 103 cells/mL in the replicates.

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
see table

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: see table
- Photoperiod: 24 h/d light
- Light intensity and quality: 60 - 120 µE • m-2 • s-1

EFFECT PARAMETERS MEASURED
Recovery of algae: After 72 h algae were transferred from test concentrations
72.0 – 450 mg/L and control into fresh untreated medium
(0.5 – 1 mL algae suspension from each replicate were filled up with dilution water to 10 mL) and allowed to grow for further
4 - 12 d to determine whether the effect of the item is reversible.

TYPE AND FREQUENCY OF MEASUREMENT: Cell density was measured via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. The cell density was measured at the beginning of the test (after application) and every 24 h. Dilution water was used as background signal. The pH-value at the beginning of the test was measured out of one additional replicate of each test concentration and control. At the end it was measured from one pool per concentration of all replicates. The room temperature was measured continuously by a hygrothermograph. Light intensity was measured prior to test start. Microscopic evaluation of the cells at the start and at the end of the incubation was determined. Cells were checked for any unusual cell shapes, colour differences, chloroplast morphology, flocculation, adherence of algae to test containers or aggregation of alga cells.

COURSE OF THE STUDY:
Based on the results of a preliminary range finding test (non GLP), a definitive test was performed with 5 concentration levels in a geometrical series with a factor 2.5 and 3 replicates each. A control with dilution water (without test item, six replicates) was tested under the same conditions as the test group. A three day-old preculture incubated at study conditions was used for the main study. Chlorophyll-fluorescence was determined at the beginning and after 24, 48 and 72 h. Recovery of algae was carried out as specified above. Other measurements were performed as specified above.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study
- Test concentrations: 10, 100 and 500 mg/l (nominal)
- Results used to determine the conditions for the definitive study: 500 mg/l 100 % Specific growth rate inhibition, 100 % Specific yield inhibition
100 mg/l: 24 % Specific growth rate inhibition, 71 % Specific yield inhibition
Reference substance (positive control):
yes
Remarks:
Potassium dichromate p.a.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
45.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 43.4 - 47.4 mg/l
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
53.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 51.1 - 56.1 mg/l
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
70.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 67.1 - 73.6 mg/l
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
68.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 67.3 - 70.2 mg/l
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
77.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 75.6 - 78.4 mg/l
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
94.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 92.3 - 95.6 mg/l
Details on results:
After 72 h algae were transferred from nominal concentrations 72.0 - 450 mg/L and control to fresh untreated medium and allowed to grow for further 4 - 12 d under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 450 mg/L (highest test concentration).
Results with reference substance (positive control):
The toxicity of potassium dichromate to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 h from March 10 to 13, 2008

EC50 Values of the Reference Item based on nominal concentrations mg/L, (0-72 h):

Yield inhibition
EyC50 0.31
95 % confidence interval 0.30 – 0.33
Rate-related inhibition
ErC50 0.74
95 % confidence interval 0.70 – 0.78

Reported statistics and error estimates:
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures according to CLOPPER and PEARSON (1934).
see attached document


EC-Values (0-72 h) and 95 % Confidence Intervals ofDimethylaminopropyl methacrylamide

based on nominal concentrations of the test item [mg/L]

 

Inhibition of yield

EyC10

45.4 (43.4 – 47.4)

EyC20

53.3 (51.1 – 56.1)

EyC50

70.5 (67.1 – 73.6)

 

Rate-related inhibition

ErC10

68.9 (67.3 – 70.2)

ErC20

77.3 (75.6 – 78.4)

ErC50

94.2 (92.3 – 95.6)

Cell Densities

 

Nominal test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

450

1

1928

0

0

1140

 

2

1928

0

0

760

 

3

1928

0

0

760

 

Mean

1928

0

0

887

 

180

1

1928

1612

1014

1140

 

2

1928

2285

1427

1520

 

3

1928

1550

394

760

 

Mean

1928

1816

945

1140

 

72.0

1

1928

7568

56948

266416

 

2

1928

8651

55721

261737

 

3

1928

8120

66533

261454

 

Mean

1928

8113

59734

263202

 

28.8

1

1928

12551

112721

572992

 

2

1928

14150

126037

535238

 

3

1928

14855

104600

539607

 

Mean

1928

13852

114453

549279

 

11.5

1

1928

17831

115412

570768

 

2

1928

13586

108084

489280

 

3

1928

13530

126527

588061

 

Mean

1928

14982

116674

549370

 

Control

1

1928

12583

93071

501528

 

2

1928

17958

98921

535424

 

3

1928

14840

113040

497159

 

4

1928

14690

106865

598881

 

5

1928

14144

133699

565402

 

6

1928

12814

118081

628579

 

Mean

1928

14505

110613

554496

 

Evaluation after 72 h

Nominal test item concentration

Replicate

Yield

Inhibition of yield

Growth rate

Rate-related inhibition

[mg/L]

No.

 

[%]

[d-1]

[%]

450

1

 

-788

100.00

 

-0.175

100.00

2

 

-1168

100.00

 

-0.310

100.00

3

 

-1168

100.00

 

-0.310

100.00

Mean

 

-1041

100.00

 

-0.265

100.00

180

1

 

-788

100.00

 

-0.175

100.00

2

 

-408

100.00

 

-0.079

100.00

3

 

-1168

100.00

 

-0.310

100.00

Mean

 

-788

100.00

 

-0.188

100.00

72.0

1

 

264488

52.13

 

1.643

12.89

2

 

259809

52.98

 

1.637

13.20

3

 

259526

53.03

 

1.637

13.22

Mean

 

261274

52.71

 

1.639

13.10

28.8

1

 

571064

-3.35

 

1.898

-0.65

2

 

533310

3.49

 

1.875

0.56

3

 

537679

2.69

 

1.878

0.41

Mean

 

547351

0.94

 

1.884

0.11

11.5

1

 

568840

-2.94

 

1.897

-0.58

2

 

487352

11.80

 

1.845

2.14

3

 

586133

-6.07

 

1.907

-1.11

Mean

 

547442

0.93

 

1.883

0.15

Control

1

 

499600

 

 

1.854

 

2

 

533496

 

 

1.876

 

3

 

495231

 

 

1.851

 

4

 

596953

 

 

1.913

 

5

 

563474

 

 

1.894

 

6

 

626651

 

 

1.929

 

Mean

 

552568

 

 

1.886

 

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

1.876

2.001

1.684

1.854

0.160

8.61

14.5

2

2.232

1.706

1.689

1.876

0.309

16.5

3

2.041

2.030

1.481

1.851

0.320

17.3

4

2.031

1.984

1.723

1.913

0.166

8.66

5

1.993

2.246

1.442

1.894

0.411

21.7

6

1.894

2.221

1.672

1.929

0.276

14.3

 

 

 

Mean

1.886

 

 

 

 

 

SD ±

0.03

 

 

 

 

CV [%]

1.68

 

SD = Standard deviation    CV = percent coefficient of variation

 

  Recovery of Alga Cells

After 72 h of exposure, 0.5 – 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from nominal test item concentrations 72.0 - 450 mg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 4 - 12 days under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 450mg/L.

Nominal test item concentration
[mg/L]

Incubation time                   [d]

Cell density [cells/mL]

Growth rate after incubation time [d-1]

Initial#

Final

450

12

89

636

0.16

624

0.16

831

0.19

180

6

114

718654

1.46

638343

1.44

537265

1.41

72.0

4

13160

1739808

1.22

1743720

1.22

1651839

1.21

Control

27725

1466497

0.99

1624581

1.02

#) calculated from the cell density of the definitive test after 72 h

Validity criteria fulfilled:
yes
Remarks:
The study meets the validity criteria of the guideline: cell growth, test temperature, difference temperature from mean, pH value in the control, mean coefficients of variation of average spec. growth rates were fulfilled.
Conclusions:
In this study Dimethylaminopropyl methacrylamide was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of yield (EyC50) and specific growth rate (ErC50) after 72 h were 70.5 (67.1 – 73.6) and 94.2 (92.3 – 95.6) mg/L, respectively.
The test effect was observed to be reversible at all tested concentrations following 72 h exposure and 4 - 12 days of growth under test conditions.
All effect levels are given based on nominal concentrations of Dimethylaminopropyl methacrylamide.
Executive summary:

In a 72 hour acute toxicity study, the cultures of Desmodesmus subspicatus were exposed to Dimethylaminopropyl methacrylamide at nominal concentrations of 11.5, 28.8, 72.0, 180 and 450 mg/L under static conditions in accordance with OECD 201. The EC10, EC20 and EC50values based on growth rate were measured.

After 72 h algae were transferred from nominal concentrations 72.0 - 450 mg/L and control to fresh untreated medium and allowed to grow for further 4 - 12 d under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 450 mg/L (highest test concentration).

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for Alga, Growth inhibition test with Desmodesmus subspicatus toxicity study.

 

Results Synopsis

Test Organism: Desmodesmus subspicatus

Test Type: Static 

72 hr ErC50:  94.2 mg/l (95% Confidence interval: 92.3 - 95.6 mg/l)  

72 hr ErC10:  68.9 mg/l (95% Confidence interval: 67.3 - 70.2 mg/l)  

  

            

Description of key information

The ErC50/ ErC10 value based on growth rate was 94.2/ 68.9 mg/L and EbC50 value based on yield was 70.5 mg/L after 72 hours,

respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
94.2 mg/L
EC10 or NOEC for freshwater algae:
68.9 mg/L

Additional information

There is one ecotoxicity study in freshwater algae available for Dimethylaminoproyl methacrylamide, which is considered reliable without restriction (Klimisch score 1; DR.U.NOACK-LABORATORIEN, 2009).

In an 72 hour acute toxicity study (static dose-response test), the cultures of Desmodesmus subspicatus strain no. CHODAT SAG 86.81 were exposed to Dimethylaminopropyl methacrylamide at nominal concentrations of 0, 11.5, 28.8, 72.0, 180, and 450 mg/L under static conditions in accordance with the guideline OECD 201. The ErC10value based on growth rate was 68.9 mg/L after 72 hours, respectively