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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2010-06-11 to 2010-07-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
An aliquot of 10 mL of the treatment and control samples were diluted with 10 mL methanol giving a sample preparation factor of two before they were deep-frozen and protected from light until analysis was performed.
Vehicle:
no
Details on test solutions:
In order to assess the toxicity of Partially unsaturated IQAC, DMS quaternised containing different components, water accommodated fractions (WAFs) with the loading rates of 0.010, 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L were tested. Additionally, a control (test water without test item) was tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
For preparation of the WAFs with the two highest loading rates of 3.2 and 10 mg/L, individual dispersions of the test item were prepared. The dispersions were stirred for 3 hours to dissolve a maximum amount of the different components of the test item in the dispersion. Then, the dispersions were filtered through membrane filters (0.45 μm) and the undiluted filtrates were tested as WAFs. Due to technical reasons, the WAFs with the lower loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L were prepared as dilutions of the WAF with the loading rate of 3.2 mg/L.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata, green alga,Strain No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen, Germany).
- Method of cultivation: according to OECD 201 ACCLIMATION
- Culturing media and conditions: An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (= 24 mg/L as CaCO3)
Test temperature:
22 °C
pH:
At the start of the test, the pH measured in the treatments was between 8.1 and 8.2. At the end of the test, pH values of 8.2 to 8.9 were measured.
Nominal and measured concentrations:
(WAFs) loading rates: 0.010, 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L

At the start of the test, the measured test item concentrations (based on two main components of the test item) in the test media with the loading rates of 0.010, 0.032, 0.10, 0.32, 1.0, 3.2, 10 mg/L were measured concentrations at the loading rates of 0.10, 0.32, 1.0, 3.2, 10 mg/L were 2.2, 6.2, 11, 14 and 22 μg/L, respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks covered with a glass dish
- Material, size, headspace, fill volume: 15 mL of test solution
- Aeration: no
- Initial cells density: 10 000 cells /mL
- Control end cells density: no data
- No. of vessels per concentration (replicates): 3
- 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: no data
- Light intensity and quality: 6970 to 8270 Lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter (Coulter Counter®, Model ZM)
- Algal biomass in the samples was determined by fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800), in duplicate.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: water accommodated fractions (WAFs) with the loading rates of 0.010, 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L were tested
- Range finding study: yes
- Results used to determine the conditions for the definitive study: The enlarged spacing factor of 3.2 between the loading rates was chosen, as according to the results of the range-finding test the concentration-effect relationship was rather flat and thus a large range had to be tested
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.8 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water accommodated fractions (WAFs)
Basis for effect:
growth rate
Remarks on result:
other: 4.3-5.5 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water accommodated fractions (WAFs)
Basis for effect:
growth rate
Remarks on result:
other: no data
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water accommodated fractions (WAFs)
Basis for effect:
growth rate
Remarks on result:
other: no data
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.5 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water accommodated fractions (WAFs)
Basis for effect:
other: yield
Remarks on result:
other: 1.2-2.0 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.032 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water accommodated fractions (WAFs)
Basis for effect:
other: yield
Remarks on result:
other: no data
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
other: water accommodated fractions (WAFs)
Basis for effect:
other: yield
Remarks on result:
other: no data
Details on results:
- Exponential growth in the control (for algal test): yes, biomass increased by a factor of 99 over 72 hours
- Observation of abnormalities: The shape and size of the algal cells were obviously not affected by the test item up to at least measured concentration.
No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

Duration Endpoint Effect conc. Nominal/Measured Conc. based on Basis for effect Remarks (e.g. 95% CL)

72 h EC10 2.1 mg/L nominal other: water accommodated fractions (WAFs) growth rate 1.7-2.5 mg/L
72 h EC20 2.9 mg/L nominal other: water accommodated fractions (WAFs) growth rate 2.6 – 3.3 mg/L
72 h EC50 4.8 mg/L nominal other: water accommodated fractions (WAFs) growth rate 4.3-5.5 mg/L
72 h NOEC 0.32 mg/L nominal other: water accommodated fractions (WAFs) growth rate no data
72 h LOEC 1 mg/L nominal other: water accommodated fractions (WAFs) growth rate no data
72 h EC10 0.22 mg/L nominal other: water accommodated fractions (WAFs) other: yield 0.11-0.35 mg/L
72 h EC20 0.43 mg/L nominal other: water accommodated fractions (WAFs) other: yield 0.26-0.61 mg/L
72 h EC50 1.5 mg/L nominal other: water accommodated fractions (WAFs) other: yield 1.2-2.0 mg/L
72 h NOEC 0.032 mg/L nominal other: water accommodated fractions (WAFs) other: yield no data
72 h LOEC 0.1 mg/L nominal other: water accommodated fractions (WAFs) other: yield no data
72 h EC10 31 µg/L meas. (geom. mean) other: water accommodated fractions (WAFs) growth rate 27-34 µg/L
72 h EC20 39 µg/L meas. (geom. mean) other: water accommodated fractions (WAFs) growth rate 36-42 µg/L
72 h EC50 56 µg/L meas. (geom. mean) other: water accommodated fractions (WAFs) growth rate 52-61 µg/L
72 h NOEC 7.6 µg/L meas. (geom. mean) other: water accommodated fractions (WAFs) growth rate no data
72 h LOEC 21 µg/L meas. (geom. mean) other: water accommodated fractions (WAFs) growth rate no data
72 h EC10 7.8 µg/L meas. (geom. mean) other: water accommodated fractions (WAFs) other: yield 4.2-11 µg/L
72 h EC20 12 µg/L meas. (geom. mean) other: water accommodated fractions (WAFs) other: yield 7.6-15 µg/L
72 h EC50 27 µg/L meas. (geom. mean) other: water accommodated fractions (WAFs) other: yield 22-32 µg/L
72 h NOEC 0.52 µg/L meas. (geom. mean) other: water accommodated fractions (WAFs) other: yield no data
72 h LOEC 2.8 µg/L meas. (geom. mean) other: water accommodated fractions (WAFs) other: yield no data

Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of the average growth rate and their 95% confidence intervals were calculated by Weibull Analysis using linear maximum likelihood regression and the WAF with the loading rate of 0.010 mg/L was excluded from Analysis to improve the fit of the regression . The 72-hour EC10, EC20 and EC50 values for the inhibition of yield and their 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression. For the determination of the LOEC and NOEC, average growth rate and yield at the test concentrations were compared to the control values by Williams t-test or Welch t-test.

Biomass of Algae

Loading

rates

(mg/L)

Mean measured**

concentrations

(μg/L)

Rep.

no.

 

Biomass of algae*

 

24 hours

48 hours

72 hours

 

Control

 

---

Mean

7.7

36.7

203.9

SD

1.0

2.9

3.3

 

0.010

 

0.31

Mean

7.8

38.1

198.4

SD

0.5

1.1

1.6

 

0.032

 

0.52

Mean

7.8

39.9

199.5

SD

0.3

1.5

2.6

 

0.10

 

2.8

Mean

5.9

32.6

160.7

SD

0.5

1.2

2.9

 

0.32

 

7.6

Mean

6.9

33.1

177.6

SD

0.4

1.2

2.3

 

1.0

 

21

Mean

6.4

30.8

133.3

SD

0.1

2.9

0.4

 

3.2

 

42

Mean

2.5

11.5

72.1

SD

0.2

0.5

0.4

 

10

 

95

Mean

0.0

0.3

0.5

SD

0.1

0.1

0.1

 SD: Standard deviation

 *: The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 104). At the start of the test, the initial cell density was 10000 algal cells/mL, corresponding to 2.06 x 103relative fluorescence units.

 **: based on two main components

 

Average Growth Rates (μ)

 

Loading

rates

(mg/L)

 

Mean measured

concentrations

(μg/L)

 

Average growth rateμ(day-1) and inhibition ofμ(Ir)

 

24 hours

48 hours

72 hours

μ

Ir(%)

μ

Ir(%)

μ

Ir(%)

 

Control

 

---

 

1.31

 

0.0

 

1.44

 

0.0

 

1.53

 

0.0

 

0.010

 

0.31

 

1.33

 

-1.2

 

1.46

 

-1.4

 

1.52 (*)

 

0.6

 

0.032

 

0.52

 

1.33

 

-1.7

 

1.48

 

-3.0

 

1.52 (*)

 

0.5

 

0.10

 

2.8

 

1.05*

 

19.6

 

1.38

 

4.0

 

1.45 (*)

 

5.2

 

0.32

 

7.6

 

1.21

 

7.4

 

1.39

 

3.5

 

1.49 (*)

 

3.0

 

1.0

 

21

 

1.14*

 

12.8

 

1.35

 

6.1

 

1.39*

 

9.2

 

3.2

 

42

 

0.20*

 

84.4

 

0.86*

 

40.3

 

1.19*

 

22.6

 

10

 

95

 

-4.17*

 

418.4

 

-1.05*

 

172.8

 

-0.47*

 

130.6

*: mean value statistically significantly lower than in the control (according to Welch t-test, one-sided smaller,α= 0.05)

(*): statistically significantly different from the control due to very low variability of control replicates, however not estimated as a biologically relevant toxic effect

 Note: Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

 

Yield (Y)

 

Loading

rates

(mg/L)

 

Mean measured

concentrations

(μg/L)

 

Yield Y (x 103) and inhibition of Y (Iy)

24 hours

48 hours

72 hours

Y

Iy(%)

Y

Iy(%)

Y

Iy(%)

 

Control

 

---

 

5.6

 

0.0

 

34.7

 

0.0

 

201.8

 

0.0

 

0.010

 

0.31

 

5.7

 

-1.5

 

36.1

 

-4.0

 

196.4 (*)

 

2.7

 

0.032

 

0.52

 

5.7

 

-2.2

 

37.9

 

-9.3

 

197.5 (*)

 

2.2

 

0.10

 

2.8

 

3.9**

 

31.4

 

30.6*

 

11.8

 

158.6*

 

21.4

 

0.32

 

7.6

 

4.9

 

13.3

 

31.0*

 

10.4

 

175.5*

 

13.0

 

1.0

 

21

 

4.4

 

22.0

 

28.8*

 

17.0

 

131.2*

 

35.0

 

3.2

 

42

 

0.5**

 

91.6

 

9.4*

 

72.9

 

70.0*

 

65.3

 

10

 

95

 

-2.0**

 

135.8

 

-1.8*

 

105.2

 

-1.5*

 

100.8

 *: mean value statistically significantly lower than in the control (according to Williams t-test, one-sided smaller,α= 0.05)

 **: mean value statistically significantly lower than in the control (according to Welch t-test, one-sided smaller,α= 0.05)

 (*): mathematically significantly different from the control due to very low variability of control replicates, however not estimated as a biologically relevant toxic effect

 Note: Percentage inhibition values in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

Validity criteria fulfilled:
yes
Remarks:
The validity criterion of increase of biomass, the mean coefficient of variation of the daily growth rates in the control and coefficient of variation of the average specific growth rates in the replicates of the control were fulfilled.
Conclusions:
In a Alga, Growth Inhibition Test accordig to OECD 201 with Pseudokirchneriella subcapitata exposed to Partially unsaturated IQAC, DMS quaternised, the test item had a statistically significant inhibitory effect on the growth rate of the algae at all test item treatments after the test period of 72 hours. However, the 72-hour LOEC for the growth rate was determined to be 1.0 mg/L (corresponding to a mean measured concentration of 21 μg/L) since at smaller loading rates the inhibition of the growth rate was well below 10% and no dose-response relationship was given. The 72-hour NOEC for the growth rate was determined to be 0.32 mg/L (corresponding to a mean measured concentration of 7.6 μg/L). The yield of the algae was also statistically significantly inhibited at all test item treatments after the test period of 72 hours. However, the 72-hour LOEC was determined to be 0.10 mg/L (corresponding to a mean measured concentration of 2.8 μg/L) since at smaller loading rates the inhibition of yield was well below 10%. The 72- hour NOEC for yield was determined to be 0.032 mg/L (corresponding to a mean measured concentration of 0.52 μg/L).
Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to Partially unsaturated IQAC, DMS quaternised at nominal concentrations of (0.010, 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L) under static conditions in accordance with the according to OECD Guideline 201 (2006). The 72-hour LOEC for the growth rate was determined to be 1.0 mg/L (corresponding to a mean measured concentration of 21 μg/L) and the 72-hour NOEC for the growth rate was determined to be 0.32 mg/L (corresponding to a mean measured concentration of 7.6 μg/L) based on water accommodated fractions (WAFs). The 72-hour LOEC for yield was determined to be 0.10 mg/L (corresponding to a mean measured concentration of 2.8 μg/L) and the 72- hour NOEC for yield was determined to be 0.032 mg/L (corresponding to a mean measured concentration of 0.52 μg/L) based on water accommodated fractions (WAFs).

The measured concentrations in the test media of the two lowest test item treatments with loading rates of 0.010 and 0.032 mg/L were <LOQbio (limit of quantification, 0.614 μg/L). The measured concentrations at the loading rates of 0.10, 0.32, 1.0, 3.2, 10 mg/L were 2.2, 6.2, 11, 14 and 22 μg/L, respectively. The biological results were related to the loading rates of the test item and to mean measured test item concentrations.

There were no compound related phytotoxic effects.

Results Synopsis

Test Organism: Pseudokirchneriella subcapitata

Test Type: Static

Growth

72hr EC10: 2.1 mg/L (31 μg/L measured) 95% C.I.: 1.7 to 2.5 mg/L (27 to 34 μg/L measured)

72hr EC20: 2.9 mg/L (39 μg/L measured) 95% C.I.: 2.6 to 3.3 mg/L (36 to 42 μg/L measured)

72hr EC50: 4.8 mg/L (56 μg/L measured) 95% C.I.: 4.3 to 5.5 mg/L (52 to 61 μg/L measured)

72hr NOEC: 0.32 mg /L (7.6 μg/L measured)

72hr LOEC: 1.0 mg /L (21 μg/L measured)

Yield

72hr EC10: 0.22 mg/L (7.8 μg/L measured) 95% C.I.: 0.11 to 0.35 mg/L (4.2 to 11 μg/L measured)

72hr EC20: 0.43 mg/L (12 μg/L measured) 95% C.I.: 0.26 to 0.61 mg/L (7.6 to 15 μg/L measured)

72hr EC50: 1.5 mg/L (27 μg/L measured) 95% C.I.: 1.2 to 2.0 mg/L (22 to 32 μg/L measured)

72hr NOEC: 0.032 mg /L (0.52 μg/L measured)

72hr LOEC: 0.10 mg /L (2.8 μg/L measured)

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010-05-25 to 2010-09-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Sampling: Samples were taken from the stock solution (2 mg/L) and the highest test concentration (0.4 mg/L) at test initiation. 2 x 50 mL samples per concentration were frozen at – 20 °C. At test end the three additionally prepared test vessels of the stock solution without algae and the vessels of the highest test concentration were frozen for chemical analysis.
Vehicle:
no
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata, green alga
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG.
- Age of inoculum (at test initiation):
- Method of cultivation: according to OECD 201

ACCLIMATION
- Culturing media and conditions (same as test or not): The stock cultures were maintained fulfilling the criteria of the OECD guidelines. Three days prior to testing a pre-culture was established in OECD growth medium to obtain exponentially growing algae for the test.
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Test temperature:
21.9 - 22.5 °C
pH:
test cultures at start: 7.78 - 7.93
test cultures at the end:7.92 - 7.99
controls during test: 7.81 - 7.91
Nominal and measured concentrations:
nominal: 79, 119, 178, 267 and 400 μg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical glass flasks covered with silicone-sponge caps
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 mL
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): static
- Initial cells density: 10 000 cells /mL
- Control end cells density: 37 197 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 8
- No. of vessels per vehicle control (replicates):

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: according to Guideline OECD 201
- Light intensity and quality: 7456-7901 Lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter (CASY 1 Model TT, Schärfe System, Reutlingen, Germany)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Test concentrations: nominal: 79, 119, 178, 267 and 400 μg/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 400 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: no data
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
277 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 239 - 303 µg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
178 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
356 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 320 - 413 µg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
181 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL: 126 - 217 µg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
79 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes/no
- Observation of abnormalities (for algal test): Microscopy revealed normal appearances of the algae cells.
Reported statistics and error estimates:
- The evaluation of the concentration-effect-relationships and the calculations of effect concentrations were based on the nominal concentrations of the test item at test start.
- The mean value of the cell counts for each concentration plot was used for plotting growth curves.
- Calculation of the percent inhibition of growth rate [r] and yield [y] was performed according to the guidelines.
- Since there was no concentration dependent inhibition the EC50 could not be determined.
- The NOEC and LOEC values for growth rate and yield were determined by the Williams Multiple Sequential t-test using a computer programme( ToxRat® Professional 2.10 Vers. 5. ToxRat® Solutions GmbH, Naheweg 15, D-52477 Alsdorf (http://www.toxrat-solutions.de).

Table1:         Percent inhibition of growth rate and yield by the test item compared to controls after 72 h.

Test item,
nominal [µg/L]

% Inhibition
of growth rate

% Inhibition
of yield

 79

2.1

7.7

119

2.8

9.9

178

2.7

9.5

267

8.1

25.4

400

25.1

61.1

 

Table 2:         Light intensities and temperature during the test

Date

Test duration

Light [lux]

Temperature [°C]

May 25, 2010

Test start

7456

22.5

May 26, 2010

day 1

7711

22.0

May 27, 2010

day 2

7736

21.9

May 28, 2010

day 3

7901

22.0

 

Table 3:        pH-values at test start and end of test

 

 

Test item concentration [µg/L]

 

Control

79

119

178

267

400

Test start

7.81

7.93

7.92

7.93

7.91

7.78

Test end

7.91

7.92

7.95

7.99

7.96

7.95

 

Validity criteria fulfilled:
yes
Remarks:
The alga growth inhibition test fulfils the validity criteria of OECD 201 (2006): - The cell number in the control cultures increased by a factor of 37.2 within the test period of 72 h (validity criterion: > 16). - Evaluation of the sectional growth rate
Conclusions:
In a Alga, Growth Inhibition Test accordig to OECD 201 with Pseudokirchneriella subcapitata exposed to the test item the respective 72h EC50 values for growth rate and yield were calculated to be > 400 µg a.i../L (highest test concentration) and 356 µg test mat./L. The 72h EC10values were calculated to be 277 and 181 µg a.i./L for growth rate and yield, respectively. The NOEC values for growth rate and yield were estimated to be 178 and 79 µg a.i./L. However, at the LOEC concentration of 267 µg/L for effects on growth rate the inhibition was only 8.1% compared to the controls.This value was statistically significant due to the low variability of the control replicates (1.5 % CV).Since effects below 10 % are generally not considered to be ecotoxicologically relevant, it is recommended to use the growth rate EC10of 277 µg a.i./L as relevant endpoint for the long-term aquatic risk assessment.
Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to the test item at nominal concentrations of 79, 119, 178, 267, 400 µg a.i./L under static conditions in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test).  The EC50values based on growth rate and yield arecalculated to be > 400 µg a.i./L (highest test concentration) and 356 µg a.i./L. The EC10values were calculated to be 277 and 181 µg a.i./L for growth rate and yield, respectively.The NOEC values for growth rate and yield were estimated to be 178 and 79 µg a.i./L.However, at the LOEC concentration of 267 µg a.i./L for effects on growth rate the inhibition was only 8.1% compared to the controls.This value was statistically significant due to the low variability of the control replicates (1.5 % CV).Since effects below 10 % are generally not considered to be ecotoxicologically relevant, it is recommended to use the growth rate EC10of 277 µg a.i./L as relevant endpoint for the long-term aquatic risk assessment.

 

No cell abnormalities were noted: 

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for OECD Guideline 201 (Alga, Growth Inhibition Test) toxicity study.

 

Results Synopsis

 

Test Organism:

Test Type: Static:

 

72 hr ErC50:  >400 μg a.i./L                 95% CL  not applicable

72 hr ErC10: 277 μg a.i./L                    95% CL:  239 - 303 µg a.i./L

72.hr NOECr:  178 μg a.i./L               Probit Slope:  -2.01

 

72 hr EyC50:  356μg a.i./L                   95% CL: 95% CL: 320 - 413 µg a.i./L

72 hr EyC10: 181 μg a.i./L                   95% CL:  95% CL: 126 - 217 µg a.i./L

72.hr NOECy:  79 μg a.i./L                 Probit Slope:  -3.343

 

Endpoint(s) Effected:  growth rate, yield
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
guideline study with restrictions: poor description of experimental details
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
For the definitive test, six concentrations of Varisoft 3690, each in triplicate, were
prepared in 20 mL silanised glass scintiallation vials containing 6 mL algal culture
medium (with EDTA). Triplicate controls in algal culture medium were also prepared.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: Printz
- Source (laboratory, culture collection): American Type Culture Collection (ATCC 22662)
- Age of inoculum (at test initiation): 4-7 days
- Method of cultivation: no data

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 ± 2°C
pH:
7.1 - 9.1
Details on test conditions:
- Test type: Static
- Temperature: 24 ± 2°C
- Light quality: "Cool white" fluorescent lighting
- Light intensity: 4000 ± 10% lux
- Photoperiod: Continuous illumination
- Test chamber size: 20 mL
- Test solution volume: 6 mL
- Renewal oftest solutions: None
- Age oftest organisms: 4 - 7 days (in exponential phase of growth)
- Initial cell density: 19.000 cells/mL
- No. replicate chambers concentration: 3
- Shaking rate: 100 rpm
- Dilution water: Algal culture medium (with EDTA)
- pH range: 7.1 - 9.1
- Dilution factor: 0.5 or 0.3
- Test duration: 72 h
- Effect measured: Growth (cell division) inhibition
- Test acceptability: At least 4x10E+5 cells/mL in the controls after 72 h. Variability in the controls not to exceed 20%.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
330 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CL: 269 - 411 µg/L
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
63 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
30 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

The mean control growth rate was 2.37 doublings per day, with a coefficient of variation of


1.8%, indicating test acceptability. The pH in the test vials ranged from 7.1-9.1 over the 72-h test.


 






















































Varisoft 3690, mg/L



Algal Cell Division rate



Mean Cell Division rate, %



Coeficient of Variation,%



Control



2.37



100



1.8



0.01



2.44



103



3.8



0.03



2.37



100



1.5



0.063



2.05



86



8.7



0.125



2.05



86



2.9



0.25



1.57



66



8.8



0.5



0.7



30



4.3



 


Oleic-acid based IQAC, DMS quaternised was very toxic to the cell division rate of the green alga Selenastrum capricornutum,with a 72-h EC50 of 0.332 mg/L (95 % confidence limits of 0.269 - 0.411ppm). Concentrations of the test item as low as 0.063 mg/L significantly inhibited algal growth rate i.e. the LOEC was 0.063 ppm. There was no observable effect (NOEC) on algal growth rate at concentrations of 0.03 ppm.


 


The acute toxicity of the test item to the golden orfe fish (Leuciscus idus) and to the water flea Daphnia magna after a 96 and 48 h exposure respectively, has been reported. HilI (1983) found that the concentration of oleic -acid based IQAC, DMS quaternised to kill 50% of golden orfe over 96 h (i.e LC50) was 2.4 mg/L, with no effect at 1.8 mg/L. Daphnia was much more sensitive, with a 48-h EC50 of 0.087 mg/L and a NOEC of 0.056 mg/L (WiIIiams and Thompson, 1983).


Based on EC50 values, the test item was less toxic to the alga Selenastrum capricornutum,than to Daphnia. However, based on NOEC values, similar sensitivity of these two species was found. Varisoft 3690 was also much less toxic to fish than to algae and the water flea.

Validity criteria fulfilled:
yes
Conclusions:
Oleic-acid based IQAC, DMS quaternised was determined to be very toxic to the growth of the alga Selenastrum capricornutum, with a 72-h EC50 value of 330 µg/L. No significant effect (NOEC) on growth of the green alga was observed at concentrations of 30 µg/L or less.
Executive summary:

In a static test performed according to the OECD 201”Alga, Growth Inhibition Test” (7th June 1984), the toxicity of oleic-acid based IQAC, DMS quaternised towards algae was tested. The toxic effect was investigated by determination of the inhibition of the biomass production and the growth rate of theSelenastrum capricornutum. The test substance was very toxic to the growth of the alga Selenastrumcapricornutum, with a 72-h EC50 value of 33 µg/L. Concentrations of oleic-acid based IQAC, DMS quaternised as low as 63 µg/L significantly inhibited algal growth rate i.e. the LOEC was 63 µg/L. No


significant effect (NOEC) on growth of the green alga was observed at concentrations of 30 µg/L or less.


There was a pH deviation of more than one unit in the test solutions and the control observed.


The total test period was 72 hours and the initial algal cell density was 1.9x10E+4 cells/ml.


No no analytical monitoring was performed.


The test item used to derive the 72h NOEC contains 24% isopropanol (72749-55-4 NICNAS Report 2000). Caused by the solvent, the water solubility and presumably the bioavailability of the test substance is elevated. The better bioavailability of the substance for the test organisms is expected to result in higher toxicity. The test result for the mixture of registration substance and isopropanol is not comparable to other tests made with the pure substance. The 72h NOEC of 30 µg/L is therefore not considered for the assessment.


 


Results Synopsis


 


Test Type: static


test substance with 24% isopropanol


Growth rate:


ErC50 (72h): 330 µg/L


LOEC (72h): 63 µg/L


NOEC (72h): 30 µg/L


Endpoint(s) Effected: growth rate, yield inhibition

Description of key information

Three studies on algal growth inhibition according to OECD Guideline 201 are available.

From these studies the acute effect concentration 72h ErC50 of > 400 µg/L and 330 µg/L are available. Two studies made with the registration substance provide deviating results for the 72h NOEC of 178 µg/L and 30 µg/L . However, the test item used to derive the lower 72h NOEC contains 24% isopropanol. Caused by the solvent, the water solubility and presumably the bioavailability of the test substance is elevated. The better bioavailability of the substance for the test organisms is expected to result in higher toxicity. The test result for the mixture of registration substance and isopropanol is not comparable to the test made with the pure substance. The 72h NOEC of 30 µg/L is therefore not considered for the assessment. The 72h NOEC of 178 µg/L is considered to be more reliable. In addition a long-term Coupled OECD Confirmatory test (5 weeks) for algae under flow through conditions for the closely related partially unsaturated IQAC, DMS quaternised is available. During the 35d exposure there was no change observed in the measured density of the algae cultures exposed to the test substance doted activated sludge unit. The 35d NOEC was determined to be > 100 µg/L, which is well in tune with the 72 h NOEC of the solvent-free oleic-acid based IQAC, DME quaternised. The Coupled OECD Confirmatory test is considered to be the better model for field conditions in surface waters at the outlet of sewage treatment plants and provide a more realistic scenario of exposure of algae.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 µg/L

Additional information

In an Alga, Growth Inhibition Test according to OECD 201 with Pseudokirchneriella subcapitata exposed to the test item the respective 72h EC50 values for growth rate and yield were calculated to be > 400 µg act. ingr. /L (highest test concentration) and 356 µg act. ingr. /L. The 72h EC10 values were calculated to be 277 and 181 µg act. ingr. /L for growth rate and yield, respectively. The NOEC values for growth rate and yield were estimated to be 178 and 79 µg act. ingr. /L. However, at the LOEC concentration of 267 µg/L for effects on growth rate, the inhibition was only 8.1% compared to the controls. This value was statistically significant due to the low variability of the control replicates (1.5 % CV). Since effects below 10 % are generally not considered to be ecotoxicological relevant, it is recommended to use the growth rate EC10 of 277 µg act. ingr. /L as relevant endpoint for the long-term aquatic risk assessment (NOEC).


In a second test, the algal toxicity of Oleic-acid based IQAC, DMS quaternised was also determined to be very toxic to the growth of the alga Selenastrum capricornutum, with a 72-h EC50 value of 330 μg/L. No significant effect (NOEC) on growth of the green alga was observed at concentrations of 30 μg/L or less. The product contains 24% of isopropanol. Caused by the solvent, the water solubility and presumably the bioavailability of the test substance is elevated. The better bioavailability of the substance for the test organisms is expected to result in higher toxicity. The test result for the mixture of registration substance and isopropanol is not comparable to tests made with the pure substance. The derived NOEC is therefore not used for the assessment. In the first study the test substance is solvent-free . This study is considered to be more reliable.


In an acute test according to DIN 38 412 Part 9 with the read across substance partially unsaturated IQAC, DMS quaternised, Algae were less sensitive. The EC50 was determined to be EC50 (72 h): ca. 1800 — 4500 µg/L act. ingr. (nominal). This value is based on the biomass and therefore not used for the assessment.


In a 5 weeks long-term toxicity study in a Coupled OECD Confirmatory Test the cultures of Scenedesmus subspicatus CHODAT (new name: Desmodesmus subspicatus) were exposed to the degradation products of the closely related partially unsaturated IQAC, DMS quaternised (CAS 86088-85-9) of an activated sludge unit at 24 °C. There was no change in the measured extinction of the algae cultures exposed to the surfactant doted activated sludge unit during the test period of 5 weeks. Only minor variation was observed. The mean extinction was 0.75 E. Visual inspection of the alga cultures showed optimal green coloured cultures, an indication of high active chlorophyll. Sedimentation of algae was not observed. The culture remained stabile over 5 weeks and no negative effects were observed. In comparison the control culture was somewhat labile, so that it could be concluded that the algae culture exposed to the degradation products was slightly promoted. During the test the read across substance, partially unsaturated IQAC, DMS quaternised, is eliminated in the model activated sludge unit by > 90%. The elimination is calculated to be 97.75% taking into account the inflow into the model activated sludge unit of the test substance with a concentration of 10 mg/l, the dilution of the effluent of the elimination unit in ratio 1:1.25 and the separately determined EC0=0.1 mg/l. Thus a NOEC of >100 μg/L is calculated.


This toxicity study is classified as assignable, though only few data are reported. As a precautionary measure a NOEC of 100µg/L is choosen.


Justification for read-across:


The structural similarities between the source and the target substances presented above are the basis for the read-across hypothesis. Adequate, reliable and available scientific information indicates that the source and target substances have similar physicochemical properties, ecotoxicological and toxicity profiles and thus support the read-across hypothesis.


Both substances are UVCB substances, produced by a similar process resulting in main constituents of the same structure, varying in the degree of saturation and chain length (C16 and C18 and mainly C18, for source and target substance, respectively). Given the underlying identical generic structure (outlined in chapter 1 and 2), similar absorption following oral or dermal uptake and the same metabolic patterns are expected for source and target substance. The findings from toxicokinetic data confirm that the discussed IQAC source and target substances are only poorly absorbed after oral application and rapidly excreted. There was no tendency for accumulation of the substance in the body of the test animals.


In conclusion the results obtained from source substance (partially unsaturated IQAC, DMS quaternised) are considered a reliable source to cover endpoints of the target substance (oleic-acid based IQAC, DMS quaternised). Beyond, the dose descriptors obtained from these studies performed on the source substance are considered as an appropriate starting point for deriving the respective PNECs.


A more detailed justification for read-across is outlined in a separate document:


“Justification for read-across - toxicological information”, is attached to the endpoint summary acute toxicity and provided in chapter 13 of Technical dossier.