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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:
2015-04-19 to 2015-04-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
All test item concentrations and the control were analytically verified via LC-MS/MS at the beginning, after 24, 48 hours and at the end of the test. Samples at test start were taken from an additional replicate without algae. For the test item analysis after 24, 48 and 72 hours the test samples were taken from the pooled test replicates per concentration level and control.
- Sample storage conditions before analysis: room temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A stock solution of 10 mg test item/L was prepared with test medium (natural river water). This solution was stirred at a temperature of 40°C ± 2°C for 24 ± 1 hours. Thereafter, the saturated solution was stirred for further 24 ± 1 hours and cooled down to the temperature of the test (21 - 24°C).
5 concentration levels in a geometrical series with a separation factor of √10, prepared by diluting the stock solution of 10 mg test item/L with test medium, were tested as follows: 0.514 – 1.08 – 2.26 – 4.76 – 10.0 mg/L. As the test item is a solid with a content of ca. 96 % the test concentrations of the pure substance were: 0.493 – 1.04 – 2.17 – 4.57 – 9.60 mg pure compound/L.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Desmodesmus subspicatus CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): two days old preculture
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE•m-2•s-1 for 24 h per day. Nutrient medium Z according to LÜTTGE et al. (1994)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.5 (22.0 - 23.0) °C
pH:
start: 8.02 - 8.06; end: 7.87 - 8.01
Nominal and measured concentrations:
nominal: 0.493, 1.04, 2.17, 4.57, 9.60 mg a.i./L
measured (geom. mean): 0.0210, 0.0347, 0.133, 2.33, 8.37 mg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, provided with cotton wool plugs. The test container were pretreated with the appropriate test solutions for 72 hours under test conditions. Before the start of the exposure phase, the test container were emptied and refilled with freshly prepared test solutions.
- Fill volume: 100 mL
- Initial cells density: nominal: approximately 2 - 5 x 1E03 cells/mL; current: 4998 cells/mL
- Control end cells density: 696778 (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no, natural river water, for details see section "any other information on materials and methods"

TEST MEDIUM / WATER PARAMETERS
river water as specified in section "any other information on materials and methods"

OTHER TEST CONDITIONS
- Sterile test conditions: The natural river water was stored at 15 ± 2 °C and at - 18± 2 °C for at least 24 hours. Freezing was found to be suitable to minimize the content of vital natural algae cells of the water as well as to reduce microbial (bacterial) activity
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: 60 - 120 µE • m-2 • s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations: sqrt(10)
Range finding study
- Test concentrations: 0.096, 0.96, 9.6, 96 mg a.i./L, nominal, prepared in natural river water
- Results used to determine the conditions for the definitive study:
Growth Rate Inhibition: 100, 100, 10, -2% at 0.096, 0.96, 9.6, 96 mg a.i./L
Yield Inhibition: 100, 100, 43, -11% at 0.096, 0.96, 9.6, 96 mg a.i./L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.205 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% c.l. 0.131 - 0.881 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.407 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% c.l. 0.213 -1.12 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 0.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.252 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% c.l. 0.103 - 0.892 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at start and the end of the incubation period revealed nomorphological abnormalities.
Results with reference substance (positive control):
- Results with reference substance valid?
- ErC50: 0.586 (95% c.l. 0.522 - 0.818mg/L); valid range: 0.678 ± 0.414
- EyC50: 0.401 (95% c.l. 0.285 - 0.469), valid range: 0.319 ± 0.141
- Other: tested from 2014-10-14 to 2014-10-17
Reported statistics and error estimates:
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.
The values for NOEC/LOEC were determined by calculation of statistical significance of inhibition of growth rate and yield. As standard methods, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were 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 tests are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is 0.05. Equal Variance test failed for yield. To pass the Equal Variance test, the two highest concentrations were excluded from statistical evaluation.

Validity Criterion

Required

This study

Increase of the cell growth in the control (72 hours)

Exponentially > 16-fold corresponding to a specific growth rate of 0.92 / day

139-fold (specific growth rate 1.64 / day)

Mean coefficients of variation for section-by-section specific growth rates (days    0-1, 1-2 and 2-3) in the control cultures

< 35 %

18.3 %

Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures

< 7 %

2.22 %

Cell Densities 

Geometric mean exposure test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

8.37

1

4998

<LOQ

<LOQ

<LOQ

2

4998

<LOQ

<LOQ

<LOQ

3

4998

<LOQ

<LOQ

<LOQ

Mean

4998

n.a.

n.a.

n.a.

2.33

1

4998

3162

<LOQ

<LOQ

2

4998

3183

<LOQ

<LOQ

3

4998

4531

<LOQ

<LOQ

Mean

4998

3625

n.a.

n.a.

0.133

1

4998

17425

146721

649803

2

4998

19427

135580

605795

3

4998

24118

153443

620349

Mean

4998

20323

145248

625316

0.0347

1

4998

20761

102154

407455

2

4998

20237

98806

360588

3

4998

21904

105312

478535

Mean

4998

20967

102091

415526

0.0210

1

4998

24507

169864

484816

2

4998

22220

191123

667475

3

4998

27388

139322

562610

Mean

4998

24705

166770

571634

Control

1

4998

22589

152625

643002

2

4998

17044

126423

582664

3

4998

24252

211299

777582

4

4998

19444

147440

685884

5

4998

24395

143190

735307

6

4998

26387

185661

756228

Mean

4998

22352

161106

696778


Evaluation after 72 hours:Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

n.a. = not applicable

 

 

Geometric mean exposure test item concentration

[mg/L]

Replicate No.

Growth rate [d-1]

Growth rate inhibition [%]

Yield [cells/mL]

Inhibition of yield [%]

8.37

1

2

3

Mean

n.a.

n.a.

n.a.

n.a.

100

100

100

100

n.a.

n.a.

n.a.

n.a.

100

100

100

100

2.33

1

2

3

Mean

n.a.

n.a.

n.a.

n.a.

100

100

100

100

n.a.

n.a.

n.a.

n.a.

100

100

100

100

0.133

1

2

3

Mean

1.62

1.60

1.61

1.61 (-)

1

3

2

2

644805

600797

615351

620318 (-)

7

13

11

10

0.0347

1

2

3

Mean

1.47

1.43

1.52

1.47(+)

11

13

8

11

402457

355590

473537

410528 (+)

42

49

32

41

0.0210

1

2

3

Mean

1.53

1.63

1.58

1.58 (-)

11

13

8

11

479818

662477

557612

566636 (-)

31

4

19

18

Control

1

2

3

4

5

6

Mean

1.62

1.59

1.68

1.64

1.66

1.67

1.64

 

638004

577666

772584

680886

730309

751230

691780

 

 

Validity criteria fulfilled:
yes
Conclusions:
The NOEC, EC10 and EC50 of C16 Alkylamidopropyltrimethylammonium Chloride to the green alga Desmodesmus subspicatus based on growth rate were 0.0202 mg a.i./L, 0.205 mg a.i./L (95% c.l. 0.131 – 0.881 mg a.i./L) and 0.407 mg a.i./L (95% c.l. 0.213 – 1.12 mg a.i./L), respectively. All values given are based on the geometric mean exposure concentrations.
Executive summary:

In a 72 hour toxicity study according to OECD Guideline 201 (2011), the cultures of Desmodesmus subspicatus CHODAT SAG 86.81 were exposed to C16 Alkylamidopropyltrimethylammonium Chloride at nominal concentrations of 0 (control), 0.493, 1.04, 2.17, 4.57, 9.60 mg a.i./L in natural river water, corresponding to geom. mean measured concentrations of 0 (control), 0.0210, 0.0347, 0.133, 2.33, 8.37 mg a.i./L under static conditions.

The test item is known to sorb to organic and inorganic materials by different mechanisms. The sorption processes are mostly non-linear, means are concentration dependent. Due to these properties the test item is difficult to test in synthetic water (e.g. sorption to the test organisms and glass walls of the test vessels) and results from such tests depend from the test settings applied.Therefore, natural river water was usedwhich contains particulate as well as dissolved organic carbon to which the test item can sorb partially to reduce the difficulties encountered in tests with synthetic water (e.g. preventing that the test item settles onto surfaces). The sorbed fraction of the test item is difficult to extract from the test system which normally leads to low analytical recoveries at the end of the exposure. Nevertheless the test item is present in the test system and therefore available for exposure (dissolved in water and sorbed).

Additionally, the test vessels were pre-treated with the test solutions one day before the start of the respective exposure interval to saturate the glass walls with sorbed test item in the definitive test.This procedure was carried out to achieve stable exposure concentrations in the test vessels and to reduce losses of the test item by adsorption onto the glass walls.

All test concentrations and the control were analytically verified by LC-MS/MS analysis at the start of the exposure (0 hours), 24, 48 hours and the end of the exposure (72 hours).

The measured concentrations of the test item at the start of the exposure were in the range of 82 to 100 % of the nominal values. After 24 h, the measured concentrations of the test item were in the range of 22 to 76 % of the nominal values. After 48 h, the measured concentrations of the test item were in the range of < LOQ to 87 % of the nominal values and at the end of the exposure, the measured concentrations of the test item were in the range of< LOQto 96 % of the nominal values. Therefore, all values given are based on the geometric mean exposure concentrations.

The NOEC, EC10 and EC50 based on growth rate inhibition were0.0202 mg a.i./L, 0.205 mg a.i./L (95% c.l. 0.131 – 0.881 mg a.i./L) and 0.407 mg a.i./L (95% c.l. 0.213 – 1.12 mg a.i./L), respectively.

The % growth inhibition in the treated algal culture as compared to the control ranged from 2% to 100%.

The NOEC, EC10 and EC50 based on yield were0.0202 mg a.i./L, < 0.0202 mg a.i./L and 0.252 mg a.i./L (95% c.l. 0.103 – 0.892 mg a.i./L), respectively.

Microscopic evaluation of the cells at start and the end of the incubation period revealed no morphological abnormalities.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for the algal growth inhibition test in Desmodesmus subspicatus.

 

Results Synopsis

Test Organism: Desmodesmus subspicatus CHODAT SAG 86.81

Test Type: Static

 

72 hr ErC10: 0.205mg a.i./L,(95% c.l. 0.131 – 0.881 mg a.i./L)

72 hr ErC50: 0.407 mg a.i./L (95% c.l. 0.213 – 1.12 mg a.i./L)

72 hr NOEC: 0.0202 mg a.i./L

Endpoint(s) Effected:  growth rate

72 hr ErC10: < 0.0202mg a.i./L

72 hr ErC50: 0.252 mg a.i./L (95% c.l. 0.103 – 0.892 mg a.i./L)

72 hr NOEC: 0.0202 mg a.i./L

Endpoint(s) Effected:  yield

Description of key information

72 h NOEC (growth rate) = 0.0202 mg a.i./L
72 h ErC10 = 0.205 mg a.i./L (95% c.l. 0.131 – 0.881 mg a.i./L)
72 h ErC50 = 0.407 mg a.i./L (95% c.l. 0.213 – 1.12 mg a.i./L) (geom. mean measured);
Desmodesmus subspicatus, static, river water; OECD Guideline 201 (2011); RL1; GLP

Key value for chemical safety assessment

EC50 for freshwater algae:
0.407 mg/L
EC10 or NOEC for freshwater algae:
0.205 mg/L

Additional information

In a 72 hour toxicity study according to OECD Guideline 201 (2011), the cultures of Desmodesmus subspicatus CHODAT SAG 86.81 were exposed to C16 Alkylamidopropyltrimethylammonium Chloride at nominal concentrations of 0 (control), 0.493, 1.04, 2.17, 4.57, 9.60 mg a.i./L in natural river water, corresponding to geom. mean measured concentrations of 0 (control), 0.0210, 0.0347, 0.133, 2.33, 8.37 mg a.i./L under static conditions.

The test item is known to sorb to organic and inorganic materials by different mechanisms. The sorption processes are mostly non-linear, means are concentration dependent. Due to these properties the test item is difficult to test in synthetic water (e.g. sorption to the test organisms and glass walls of the test vessels) and results from such tests depend from the test settings applied.Therefore, natural river water was usedwhich contains particulate as well as dissolved organic carbon to which the test item can sorb partially to reduce the difficulties encountered in tests with synthetic water (e.g. preventing that the test item settles onto surfaces). The sorbed fraction of the test item is difficult to extract from the test system which normally leads to low analytical recoveries at the end of the exposure. Nevertheless the test item is present in the test system and therefore available for exposure (dissolved in water and sorbed).

Additionally, the test vessels were pre-treated with the test solutions one day before the start of the respective exposure interval to saturate the glass walls with sorbed test item in the definitive test.This procedure was carried out to achieve stable exposure concentrations in the test vessels and to reduce losses of the test item by adsorption onto the glass walls.

All test concentrations and the control were analytically verified by LC-MS/MS analysis at the start of the exposure (0 hours), 24, 48 hours and the end of the exposure (72 hours).

The measured concentrations of the test item at the start of the exposure were in the range of 82 to 100 % of the nominal values. After 24 h, the measured concentrations of the test item were in the range of 22 to 76 % of the nominal values. After 48 h, the measured concentrations of the test item were in the range of < LOQ to 87 % of the nominal values and at the end of the exposure, the measured concentrations of the test item were in the range of< LOQto 96 % of the nominal values. Therefore, all values given are based on the geometric mean exposure concentrations.

The NOEC, EC10 and EC50 based on growth rate inhibition were 0.0202 mg a.i./L, 0.205 mg a.i./L (95% c.l. 0.131 – 0.881 mg a.i./L) and 0.407 mg a.i./L (95% c.l. 0.213 – 1.12 mg a.i./L), respectively.

The % growth inhibition in the treated algal culture as compared to the control ranged from 2% to 100%.

The NOEC, EC10 and EC50 based on yield were 0.0202 mg a.i./L, < 0.0202 mg a.i./L and 0.252 mg a.i./L (95% c.l. 0.103 – 0.892 mg a.i./L), respectively.

Microscopic evaluation of the cells at start and the end of the incubation period revealed no morphological abnormalities.