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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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP, all validity criteria fulfilled, identification complete, including chemical analyses
Justification for type of information:
Read-across data is presented from quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808-53-2, EC Number 276-038-9. This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Cocobis(2-hydroxyethyl) methylammonium chloride (CAS No. 70750-47-9)
- Physical state: Paste-like, yellow-brownish
- Active ingredient content: 99.4%
97.9 % Cocobis(2-hydroxyethyl) methylammonium chloride
1.5 % Cocobis(2-hydroxyethyl) amine
- Purity test date: 2009-06-02
- Lot/batch No.: S001700
- Expiration date of the lot/batch: 2019-04-28
- Storage condition of test material: Room temperature, protected from moisture and light, under nitrogen gas.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: From the series of concentrations the second lowest and the second highest test concentration (2.00 and 200 µg/L) and the control were analytically verified at the beginning of the test.
- Sampling method: Separate replicates for the test item analysis after 0 and 72 h were prepared without algae. Analysis after 72 h of these concentrations were carried out. Sorption to the walls of the glass container was checked out of one test replicate (prepared with algae) of a concentration level close to the EC50-value (the second highest test concentration of 200 µg/L was analysed).
- Sample storage conditions before analysis: All samples were stored at room temperature, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was freshly prepared. Dispersion treatment was 5 min ultrasound at room temperature and an equilibration phase for 1 h before application.
- Eluate: Natural river water
- Differential loading: 0.640 - 2.00 - 6.40 - 20.0 - 64.0 - 200 - 640 µg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDAK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 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): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
After 72 h of exposure, 0.2 – 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from test concentrations 200 – 640 µg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 4 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 640 µg/L (highest test concentration).
Hardness:
Not measured
Test temperature:
Mean: 23.5 °C, Min: 23 °C, Max: 24 °C
pH:
Nominal test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
640 8.04 8.13
200 8.05 8.41
64.0 8.04 8.92
20.0 8.05 8.96
6.40 8.03 8.94
2.00 8.02 8.94
0.640 7.96 8.93
Control 7.96 8.86
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with 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: 9787 cells/mL
- Control end cells density: Mean 3370253 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: Water of the river Innerste was used. This river is located in
Sarstedt / Ruthe. Water parameters are given in Table 1. Additionally 50 % of the components concentrations of the dilution water (total application volume 6.5 mL/L) acc. to the guideline were added to enable a sufficient growth of algae
Characterisation Data of Natural River Water
River Hüpeder Bach
Location D-30982 Pattensen, Am Pfingstanger
Sampling Date 2009-10-07
Weather on Day of Sampling dry, few clouds, ca. 22 °C
Colour Yellowish, clear
pH 7.79
Conductivity [µS/cm] 531
DOC [mg C/L] 3.8
TOC [mg C/L] 3.7
Ammonium-N [mg N/L] < 0.015
Nitrate-N [mg N/L] 3.06
o-Phosphate-P [mg P/L] 0.116
Total Phosphate [mg P/L] 0.140
Suspended Matter [mg/L] 17.6
Total Hardness [mg CO3/L] 170

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)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
10.0 100 100
1.00 79 99
0.10 9 39
0.01 3 15
0.001 3 16
0.0001 3 13
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
121 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: (100 - 140)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
413.5 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: (361 - 467)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
122 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
99.4% purity
Basis for effect:
growth rate
Remarks on result:
other: (101-141)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
416 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
99.4% purity
Basis for effect:
growth rate
Remarks on result:
other: (363 - 470)
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
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.82 (CI 0.78 - 0.85) mg/L
Yield Inhibition: 0.37 (CI 0.36 - 0.40) mg/L
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 of EC10-, EC20- and EC50-values were carried out from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

Cell Densities

 

Nominal
test item concentration

Replicate

Cell density [cells/mL]

[µg/L]

No.

0 h

24 h

48 h

72 h

 

640

1

9787

26923

42819

43195

 

2

9787

27887

39327

35552

 

3

9787

25847

40149

37893

 

Mean

9787

26886

40765

38880

 

200

1

9787

30763

194187

1034288

 

2

9787

38894

218711

1128274

 

3

9787

31924

196302

976008

 

Mean

9787

33860

203067

1046190

 

 64.0

1

9787

37263

341118

2409034

 

2

9787

45582

493224

3090534

 

3

9787

39839

409108

2489357

 

Mean

9787

40895

414483

2662975

 

 20.0

1

9787

40920

554611

3216320

 

2

9787

52242

557755

3357959

 

3

9787

42551

549718

3329021

 

Mean

9787

45238

554028

3301100

 

   6.40

1

9787

39914

516884

3090252

 

2

9787

43162

514548

3255095

 

3

9787

53436

582741

3548718

 

Mean

9787

45504

538058

3298022

 

   2.00

1

9787

46936

561595

3444655

 

2

9787

49135

593311

3671411

 

3

9787

43712

499240

3377948

 

Mean

9787

46594

551382

3498005

 

   0.640

1

9787

49479

583337

2604361

 

2

9787

45187

569566

3559392

 

3

9787

47801

536544

3306339

 

Mean

9787

47489

563149

3156697

 

Control

1

9787

45606

531901

3339225

 

2

9787

41291

518045

3208875

 

3

9787

48844

587596

3425573

 

4

9787

44741

591765

3268349

 

5

9787

42034

674315

3396776

 

6

9787

49742

575103

3582722

 

Mean

9787

45376

579788

3370253

 

Evaluation after 72 h

             

Nominal
test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[µg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

640

1

0.495

74.6

33408

99.0

2

0.430

77.9

25765

99.2

3

0.451

76.8

28106

99.2

Mean

0.459

76.4

29093

99.1

200

1

1.55

20.2

1024501

69.5

2

 

1.58

18.7

1118487

66.7

3

1.53

21.2

966221

71.3

Mean

1.56

20.1

1036403

69.2

 64.0

1

1.84

 5.74

2399247

28.6

2

1.92

 1.47

3080747

 8.32

3

1.85

 5.18

2479570

26.2

Mean

1.87

 4.13

2653188

21.0

 20.0

1

1.93

 0.790

3206533

 4.58

2

1.95

 0.050

3348172

 0.370

3

1.94

 0.200

3319234

  1.23

Mean

1.94

 0.350

3291313

 2.06

   6.40

1

 

1.92

 1.47

3080465

 8.33

2

1.94

 0.580

3245308

 3.43

3

1.96

 -0.890

3538931

 -5.31

Mean

1.94

 0.390

3288235

 2.15

   2.00

1

 

1.96

 -0.380

3434868

 -2.21

2

1.98

 -1.48

3661624

 -8.96

3

1.95

 -0.050

3368161

 -0.230

Mean

1.96

 -0.640

3488218

 -3.80

   0.640

1

 

1.86

 4.40

2594574

22.79

2

1.97

 -0.950

3549605

 -5.63

3

1.94

  0.320

3296552

 1.90

Mean

1.92

  1.26

3146910

 6.35

Control

1

1.94

3329438

2

1.93

3199088

3

1.95

3415786

4

1.94

3258562

5

1.95

3386989

6

1.97

3572935

Mean

1.95

3360466

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

±

VC %

Mean

VC %

section-by-section                           

0 - 24 h

24 - 48 h

48 - 72 h

Control

1

1.54

2.46

1.84

1.94

0.468

24.1

27.4

2

1.44

2.53

1.82

1.93

0.553

28.6

3

1.61

2.49

1.76

1.95

0.470

24.0

4

1.52

2.58

1.71

1.94

0.567

29.3

5

1.46

2.78

1.62

1.95

0.719

36.9

6

1.63

2.45

1.83

1.97

0.428

21.8

Mean

1.95

 

SD ±

0.01

 

VC %

0.66

 

Validity criteria fulfilled:
yes
Conclusions:
Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides, CAS Number 70750-47-9, EC Number 274-846-6 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values (nominal concentrations): The EC10-values with 95 % confidence intervals for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 122 (101 – 141) and 39.7 (24.5 – 61.7) µg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 416 (363 – 470) and 132 (108 – 161) µg/L, respectively.
The test item effect was observed to be reversible at 200 – 640 µg/L. Therefore, there is potential for recovery following exposure up to 640 µg/L (highest test concentration).
Executive summary:

The toxicity to aquatic algae and cyanobacteria of the test substance, Quaternary ammonium compounds, coco alkylbis(hydroxyethyl)methyl chlorides, CAS Number 70750 -47 -9, EC Number 274-846-6, [Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9], was investigated in a study conducted according to OECD guideline 201 (Alga, Growth Inhibition Test).

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides, CAS Number 70750-47-9, EC Number 274-846-6 was found to inhibit the growth of the freshwater green alga, Pseudokirchneriella subcapitata, after 72 h with the following effect values (nominal concentrations): The EC10-values with 95 % confidence intervals for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 122 (101 – 141) and 39.7 (24.5 – 61.7) µg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 416 (363 – 470) and 132 (108 – 161) µg/L, respectively.

Description of key information

...........................

In a supporting study the toxicity to aquatic algae and cyanobacteria of the test substance, Quaternary ammonium compounds, coco alkylbis(hydroxyethyl)methyl chlorides, CAS Number 70750 -47 -9, EC Number 274-846-6, [Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9], was investigated in a study conducted according to OECD guideline 201 (Alga, Growth Inhibition Test).

This substance is considered to be close enough in structural integrity to the target substance, bis(2 -hydroxyethyl)oleylmethylammonium chloride, CAS Number 18448 -65 -2, EC Number 242 -332 -0, so as to justify valid read-across.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides, CAS Number 70750-47-9, EC Number 274-846-6was found to inhibit the growth of the freshwater green alga,Pseudokirchneriella subcapitata,after 72 h with the following effect values (nominal concentrations): The EC10-values with 95 % confidence intervals for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 122 (101 – 141) and 39.7 (24.5 – 61.7) µg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 416 (363 – 470) and 132 (108 – 161) µg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.414 mg/L

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