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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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From March 29, 2010 to April 15, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 as RA study
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: From the series of concentrations the second lowest and the second highest test concentration (125 and 500 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 500 µg/L was analysed).
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.
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, equilibration phase for 1 h before application was carried out.
- Eluate: NAtural river water
- Differential loading: 62.5 - 125 - 250 - 500 - 1000 µg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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:
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 500 – 1000 µg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 5 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 1000 µg/L (highest test concentration).
For details please refer to the table under information on materials and methods
Hardness:
Not measured
Test temperature:
Mean: 21.5 °C, Min: 21 °C, Max: 22 °C
pH:
Nominal test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
1000 8.02 8.01
500 8.03 8.01
250 8.06 8.37
125 8.06 8.92
62.5 8.07 9.07
Control 8.06 9.14
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to information on materials and methods
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 2742164 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 Leine was used. This river is located near D-31171 Nordstemmen, Marienbergstraße, Germany (52° 10’ 14.98‘’, 9° 46’ 7.64’’). 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
River Leine
Location D-31171 Nordstemmen, Marienbergstrasse
Sampling Date 2009-12-15
Weather on Day of Sampling cloudy, ca. - 1 °C
Colour Yellowish, clear
pH 7.97
Conductivity [µS/cm] 386
DOC [mg C/L] 3.9
TOC [mg C/L] 3.9
Ammonium-N [mg N/L] 0.042
Nitrate-N [mg N/L] 2.62
o-Phosphate-P [mg P/L] 0.062
Total Phosphate [mg P/L] 0.053
Suspended Matter [mg/L] 16.2
Total Hardness [mg CO3/L] 154


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 89 99
0.10 1 3
0.01 - 6 - 32
0.001 - 7 - 37









Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
139.8 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Remarks:
Corrected for a.i. and 45% sorption
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
269 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (200 - 351)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
200 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
103.95 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Remarks:
Corrected for a.i. and 45% sorption
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
453 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (388-514)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
235.4 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Remarks:
corrected for a.i. and 45% sorption
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
277 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
143.9 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Remarks:
corrected for a.i. and 45% sorption
Basis for effect:
other: Yield
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: 0.74 (CI 0.71 - 0.78) mg/L
Yield Inhibition: 0.36 (CI < 0.35 - 0.36) mg/L
Reported statistics and error estimates:
EC10-, EC20- and EC50-values of the growth rate and yield
analyses inhibition after 72 h were calculated by sigmoidal dose-response regression and 0 – 100 variable slope equation. 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.

EC10-, EC20-and EC50- values and 95 % Confidence Intervals of
N,N,N',N',N’’-Pentamethyl-N-tallowalkyl-1,3-propanediammonium chloride (CAS No. 68607-29-4) (0-72 h)

                                based on nominal test item concentrations [µg/L]

                               

Rate-related inhibition

ErC10

269 (200 – 351)

ErC20

322 (254 – 388)

ErC50

453 (388 – 514)

Inhibition of yield

EyC10

200 (152 – 236)

EyC20

225 (178 – 248)

EyC50

277 (254 – 375)

Cell Densities

 

Nominal
test item concentration

Replicate

Cell density [cells/mL]

[µg/L]

No.

0 h

24 h

48 h

72 h

 

1000

1

9787

23896

15906

17373

 

2

9787

21109

19972

10638

 

3

9787

19013

18628

15013

 

Mean

9787

21339

18169

14341

 

  500

1

9787

26919

36676

45220

 

2

9787

27172

32615

52557

 

3

9787

44520

74567

363923

 

Mean

9787

32870

47953

153900

 

  250

1

9787

35303

276296

1560834

 

2

9787

39524

310150

1805422

 

3

9787

42678

353094

2091520

 

Mean

9787

39168

313180

1819259

 

  125

1

9787

59410

586397

2864605

 

2

9787

68838

659571

3158989

 

3

9787

73857

749999

3237056

 

Mean

9787

67368

665322

3086883

 

    62.5

1

9787

64274

745257

3107670

 

2

9787

69585

646980

2771469

 

3

9787

78642

722533

3017782

 

Mean

9787

70834

704923

2965640

 

Control

1

9787

65675

645222

2583972

 

2

9787

60190

554785

2438766

 

3

9787

74703

806164

2544032

 

4

9787

66206

809642

3065234

 

5

9787

65402

695714

2922748

 

6

9787

71376

715299

2898233

 

Mean

9787

67259

704471

2742164

 

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]

[%]

1000

1

0.191

89.8

7586

  99.7

2

0.028

98.5

851

100

3

0.143

92.4

5226

  99.8

Mean

0.121

93.6

4554

  99.8

  500

1

0.510

72.8

35433

  98.7

2

 

0.560

70.2

42770

  98.4

3

1.21

35.8

354136

  87.0

Mean

0.76

59.6

144113

  94.7

  250

1

1.69

  9.94

1551047

  43.2

2

1.74

  7.36

1795635

  34.3

3

1.79

  4.75

2081733

  23.8

Mean

1.74

  7.35

1809472

  33.8

  125

1

1.89

 -0.84

2854818

   -4.48

2

1.93

 -2.58

3149202

 -15.3

3

1.93

 -3.01

3227269

 -18.1

Mean

1.92

 -2.14

3077096

 -12.6

    62.5

1

 

1.92

 -2.28

3097883

 -13.4

2

1.88

 -0.25

2761682

   -1.07

3

1.91

 -1.76

3007995

 -10.1

Mean

1.90

 -1.43

2955853

   -8.18

Control

1

1.86

2574185

2

1.84

2428979

3

1.85

2534245

4

1.92

3055447

5

1.90

2912961

6

1.90

2888446

Mean

1.88

2732377

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

1.90

2.29

1.39

1.86

0.450

24.2

26.3

2

1.82

2.22

1.48

1.84

0.371

20.2

3

2.03

2.38

1.15

1.85

0.634

34.2

4

1.91

2.50

1.33

1.92

0.586

30.6

5

1.90

2.36

1.44

1.90

0.465

24.5

6

1.99

2.31

1.40

1.90

0.459

24.2

Mean

1.88

 

SD ±

0.03

 

VC %

1.63

 

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the EyC50 and ErC50 (0-72 h) were 277 µg/L(equivalent to 143.9µg a.i./L) and 453µg/L (i.e., equivalent to 235.4µg a.i./L), respectively. The ErC10, EyC10, values were determined at 269 µg/L (i.e., equivalent to 139.8 µg a.i./L), and 200 µg/L (i.e., equivalent to 103.95µg a.i./L) (nominal) respectively
Executive summary:

A study was conducted to determine the toxicity of the read across substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3-propanediammonium chloride to aquatic algae, according to OECD Guideline 201, in compliance with GLP.Pseudokirchneriella subcapitatawere exposed for 72 h to nominal concentrations of62.5, 125, 250, 500 and 1000 µg/Lof the test substance.Three replicates were tested for each of the test substance concentrations and six replicates for the control. Analytical dose verification was performed. Chemical analysis of duplicate samples at125 and 500 µg/Ltaken at the beginning (64 - 67 %recovery) and at the end (25 - 31%recovery) of the test indicated biodegradation as possible reason for this decrease is very unlikely considering the short time frame. Also the river water was frozen before use to minimize the microbial activity. The decrease is attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the adsorbed fraction. Test substance recovered from the glassware (227 µg/L at concentration level 500 µg/L corresponding to 45 % of the nominal concentration). All effect values are given based on nominal concentrations of the test substance.Growth rate and biomass were recorded throughout the experiment. The EC10 for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 269 and 200 µg/L, respectively. The EC50 for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 453 and 277 µg/L, respectively.Under the study conditions, the EyC50 and ErC50 (0 -72 h) were277 µg/L(equivalent to 143.9µg a.i./L) and453µg/L (equivalent to 235.4µg a.i./L), respectively and the ErC10 and EyC10 were269µg/L (equivalent to 139.8µg a.i./L) and 200µg/L(equivalent to 103.95µg a.i./L) (nominal) respectively (Scheerbaum, 2010).

Description of key information

Key value for chemical safety assessment

EC50 for freshwater algae:
235.4 µg/L
EC10 or NOEC for freshwater algae:
139.8 µg/L

Additional information

A study was conducted to determine the toxicity of the read across substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3-propanediammonium chloride to aquatic algae, according to OECD Guideline 201, in compliance with GLP. Pseudokirchneriella subcapitata were exposed for 72 h to nominal concentrations of 62.5, 125, 250, 500 and 1000 µg/L of the test substance. Three replicates were tested for each of the test substance concentrations and six replicates for the control. Analytical dose verification was performed. Chemical analysis of duplicate samples at 125 and 500 µg/L taken at the beginning (64 - 67 % recovery) and at the end (25 - 31% recovery) of the test indicated biodegradation as possible reason for this decrease is very unlikely considering the short time frame. Also the river water was frozen before use to minimize the microbial activity. The decrease is attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the adsorbed fraction. Test substance recovered from the glassware (227 µg/L at concentration level 500 µg/L corresponding to 45 % of the nominal concentration). All effect values are given based on nominal concentrations of the test substance. Growth rate and biomass were recorded throughout the experiment. The EC10 for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 269 and 200 µg/L, respectively. The EC50 for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 453 and 277 µg/L, respectively. Under the study conditions, the EyC50 and ErC50 (0 -72 h) were 277 µg/L (equivalent to 143.9µg a.i./L) and 453 µg/L (equivalent to 235.4 µg a.i./L), respectively and the ErC10 and EyC10 were 269 µg/L (equivalent to 139.8 µg a.i./L) and 200 µg/L(equivalent to 103.95 µg a.i./L) (nominal) respectively (Scheerbaum, 2010).