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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:
24 June 2016 to 12 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
OECD guidelines for Testing of Chemicals, guideline No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", Adopted March 23, 2006; Annex 5 corrected 28 July 2011.
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Lowinox® 22IB46
Appearance: White to cream coloured powder
Batch: WB44L0016
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 18 November 2018 (retest date)
Test item: 207366/A
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: 2,2’-(2-methylpropylidene)bis[4,6-xylenol]
CAS Number: 33145-10-7
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated
Analytical monitoring:
yes
Details on sampling:
Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing undissolved residue was kept for possible analysis.
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 2.0 mL
Storage: Samples were stored in a freezer until analysis.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the highest test item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, single reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
not specified
Details on test solutions:
The batch of Lowinox® 22IB46 tested was a white to cream coloured powder with a purity of 99.7% and not completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. Thereafter, the resulting mixture was filtered over a 0.45 μm membrane filter (Whatman; RC55). The obtained Saturated Solution (SS) was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of e4 cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh water algae culture
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap water purified by reverse osmosis)
Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 50e4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).
pH:
The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 units).
Dissolved oxygen:
Not specified
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal concentrations:
Test concentrations
Lowinox® 22IB46: 1.0, 10 and 100% of a SS prepared at 100 mg/L.
Controls: Test medium without test item or other additives.
Replicates: 6 replicates of the control and the highest test concentration, 3 replicates of each intermediate test concentration, 1 extra replicate of the control and each test concentration for sampling purposes after 24 hours, 1 or 2 replicates of each concentration without algae.
Details on test conditions:
Test duration 72 hours
Test type: Static
Test vessels: 100 mL, all-glass, containing 50 mL of test solution
Medium: M2
Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 87 to 91 μE/m2/s.
Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements
pH: At the beginning and at the end of the test.
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the final test, microscopic observations were performed on the control and the highest test concentration to observe for any abnormal appearance of the algae.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.067 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate & yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.067 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate & yield
Details on results:
Measured test item concentrations
Samples taken from the highest test concentration with and without algae were analysed. At the start of the test, the measured concentrations were 0.17 and 0.18 mg/L, respectively. The concentrations did not remain stable during the test duration and were at 49-71% of initial after 24 hours of exposure and at 5.2-11% of initial at the end of the test.
Based on these results, the Time Weight Average (TWA) concentrations were calculated to be 0.067 and 0.072 mg/L in the samples with and without algae, respectively.

Inhibition of growth rate and inhibition of yield
No statistically significant differences were recorded between the values for growth rate or yield at the highest test concentration when compared to the control group.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the limit concentration when compared to the control.

Experimental conditions
The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 units). During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).
Results with reference substance (positive control):
Potassium Dichromate significantly inhibited the growth rate of this fresh water algal species at nominal concentrations of 0.56 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 1.0 mg/L with a 95% confidence interval ranging from 0.97 to 1.0 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.43 mg/L with a 95% confidence interval ranging from 0.42 to 0.44 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
Not specified in the study report.

Percentage inhibition of growth rate (total test period)

Lowinox® 22IB46

% SS prep. at 100 mg/L

Mean

Std. Dev.

n

%Inhibition

Control

1.0

10

100

1.591

1.605

1.611

1.558

0.0506

0.0378

0.0252

0.0258

6

3

3

6

 

-0.9

-1.3

2.1

 

Percentage inhibition of growth rate at different time intervals

Lowinox® 22IB46

% SS prep. at 100 mg/L

n

0 – 24 h

24 – 48 h

48 – 72 h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

100

6

6

1.363

1.335

 

2.1

1.716

1.578

 

8.0

1.693

1.760

 

-4.0

 

Percentage inhibition of yield (total test period)

Lowinox® 22IB46

% SS prep. at 100 mg/L

Mean

Std. Dev.

n

%Inhibition

Control

1.0

10

100

118.4

122.9

124.9

106.3

18.87

13.85

9.73

8.10

6

3

3

6

 

-3.7

-5.5

10.3

 

Effect parameters

Parameter1

(mg/L)

NOEC

EC10

EC20

EC50

Growth rate

Yield

0.067

0.067

>0.067

<0.067

>0.067

>0.067

>0.067

>0.067

1Time weighted average concentration in a saturated solution prepared at a loading rate of 100 mg/L.

 

pH levels recorded during the test

Lowinox® 22IB46

% SS prep. at 100 mg/L

pH

t = 0 h

t = 72 h

Control

100

8.3

8.2

7.8

7.7

 

Individual cell densities (x104cells/mL)

Time

Replicate

Lowinox® 22IB46; % SS prep. at 100 mg/L

Control

1.0

10

100

0 h

 

 

 

 

 

 

n:

Mean:

Std. Dev.:

CV:

1

2

3

4

5

6

1.000

1.000

1.000

1.000

1.000

1.000

 

6

1.0

0.0

0.0

1.000

1.000

1.000

 

 

 

 

3

1.0

0.0

0.0

1.000

1.000

1.000

 

 

 

 

3

1.0

0.0

0.0

1.000

1.000

1.000

1.000

1.000

1.000

 

6

1.0

0.0

0.0

24 h

 

 

 

 

 

 

n:

Mean:

Std. Dev.:

CV:

1

2

3

4

5

6

3.734

4.258

4.712

3.678

4.206

3.082

 

6

3.9

0.6

14.4

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

3.458

4.052

4.231

3.678

3.450

3.995

 

6

3.8

0.3

8.6

48 h

 

 

 

 

 

 

n:

Mean:

Std. Dev.:

CV:

1

2

3

4

5

6

18.366

21.627

29.080

20.492

22.056

20.312

 

6

22.0

3.7

16.8

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

18.193

16.862

18.955

18.764

16.710

21.360

 

6

18.5

1.7

9.2

72 h

 

 

 

 

 

 

n:

Mean:

Std. Dev.:

CV:

1

2

3

4

5

6

101.782

126.724

151.607

105.697

124.289

106.437

 

6

119.4

18.9

15.8

125.254

136.944

109.358

 

 

 

 

3

123.9

13.8

11.2

137.119

120.689

119.867

 

 

 

 

3

125.9

9.7

7.7

107.879

101.250

115.369

114.052

94.309

110.719

 

6

107.3

8.1

7.6

n.d.: not determined; n.a.: not applicable

According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. In the current test it was found to be 119. The test fulfils this validity criterion.

 

Individual growth rates (day-1)

Time

Replicate

Lowinox® 22IB46; % SS prep. at 100 mg/L

Control

1.0

10

100

0 – 24 h

 

 

 

 

 

 

Mean:

Std. Dev.:

n:

CV:

1

2

3

4

5

6

1.317

1.449

1.55

1.302

1.437

1.126

 

1.363

0.1483

6

10.9

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

1.241

1.399

1.442

1.302

1.238

1.385

 

1.335

0.0866

6

6.5

0 - 48 h

 

 

 

 

 

 

Mean:

Std. Dev.:

n:

CV:

1

2

3

4

5

6

1.455

1.537

1.685

1.51

1.547

1.506

 

1.54

0.0779

6

5.1

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

1.451

1.413

1.471

1.466

1.408

1.531

 

1.456

0.045

6

3.1

0 - 72 h

 

 

 

 

 

 

Mean:

Std. Dev.:

n:

CV:

1

2

3

4

5

6

1.541

1.614

1.674

1.554

1.607

1.556

 

1.591

0.0506

6

3.2

1.61

1.64

1.565

 

 

 

 

1.605

0.0378

3

2.4

1.64

1.598

1.595

 

 

 

 

1.611

0.0252

3

1.6

1.56

1.539

1.583

1.579

1.516

1.569

 

1.558

0.0258

6

1.7

n.d.: not determined; n.a.: not applicable

The coefficient of variation of the mean specified growth rate replicates in the control between 0 and 72 h was: 3.2%. According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must exceed 7%. The test fulfils this validity criterion.

 

Section-by-section growth rate

Time

Replicate

Lowinox® 22IB46; % SS prep. at 100 mg/L

Control

1.0

10

100

0 - 24 h

 

 

 

 

 

 

Mean:

Std. Dev.:

n:

CV:

1

2

3

4

5

6

1.317

1.449

1.55

1.302

1.437

1.126

 

1.363

0.1483

6

10.9

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

1.241

1.399

1.442

1.302

1.238

1.385

 

1.335

0.0866

6

6.5

24 – 48 h

 

 

 

 

 

 

Mean:

Std. Dev.:

n:

CV:

1

2

3

4

5

6

1.593

1.625

1.82

1.718

1.657

1.886

 

1.716

0.1153

6

6.7

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

1.66

1.426

1.6

1.63

1.578

1.676

 

1.578

0.0984

6

6.2

48 – 72 h

 

 

 

 

 

 

Mean:

Std. Dev.:

n:

CV:

1

2

3

4

5

6

1.712

1.768

1.651

1.641

1.729

1.656

 

1.693

0.0513

6

3

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

1.78

1.793

1.806

1.805

1.731

1.645

 

1.76

0.0625

6

3.6

n.d.: not determined; n.a.: not applicable

The mean of the replicate coefficients of variation for section-by-section growth rate in the control was: 13%. According to OECD 201, the mean coefficient of variation, measured in the control from 0 to 72 h, must not be higher than 35%. The test fulfils this validity criterion.

 

Individual yields

Time

Replicate

Lowinox® 22IB46; % SS prep. at 100 mg/L

Control

1.0

10

100

24 h

 

 

 

 

 

 

Mean:

Std. Dev.:

n:

CV:

1

2

3

4

5

6

2.7

3.3

3.7

2.7

3.2

2.1

 

2.9

0.57

6

19.3

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

2.5

3.1

3.2

2.7

2.5

3

 

2.8

0.33

6

11.7

48 h

 

 

 

 

 

 

Mean:

Std. Dev.:

n:

CV:

1

2

3

4

5

6

17.4

20.6

28.1

19.5

21.1

19.3

 

21

3.7

6

17.6

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

n.d.

n.d.

n.d.

 

 

 

 

n.a.

n.a.

n.a.

n.a.

17.2

15.9

18

17.8

15.7

20.4

 

17.5

1.7

6

9.7

72 h

 

 

 

 

 

 

Mean:

Std. Dev.:

n:

CV:

1

2

3

4

5

6

100.8

125.7

150.6

104.7

123.3

105.4

 

118.4

18.87

6

15.9

124.3

135.9

108.4

 

 

 

 

122.9

13.85

3

11.3

136.1

119.7

118.9

 

 

 

 

124.9

9.73

3

7.8

106.9

100.3

114.4

113.1

93.3

109.7

 

106.3

8.1

6

7.6

n.d.: not determined; n.a.: not applicable

 

Overview of % inhibition of growth rate in the reference test:

Nominal conc.

K2Cr2O7(mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

0.18

0.32

0.56

1.0

1.8

3.2

1.637

1.619

1.533

1.221

0.801

0.388

0.185

0.0382

0.0347

0.0494

0.0263

0.0943

0.038

0.0714

3

3

3

3

3

3

3

 

1.1

6.4

25.4

51.1

76.3

88.7

 

Overview of % inhibition of yield in the reference test:

Nominal conc.

K2Cr2O7(mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

0.18

0.32

0.56

1.0

1.8

3.2

135.5

128.0

99.2

38.1

10.4

2.2

0.8

15.16

13.04

15.28

3.03

3.43

0.04

0.40

3

3

3

3

3

3

3

 

5.5

26.8

71.9

92.3

98.4

99.4

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of Lowinox® 22IB46 tested.
The EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) were beyond the range tested, i.e. exceeded 0.067 mg/L, being the TWA concentration in a filtrate prepared at a loading rate of 100 mg/L.
The 72h-NOEC for growth rate inhibition and the 72h-NOEC for yield inhibition were at or above the limit of solubility (0.067 mg/L being the TWA concentration in a filtrate prepared at a loading rate of 100 mg/L).
Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with Lowinox® 22IB46.

 

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009 and the OECD series on testing and assessment number 23, 2000.

 

The batch of Lowinox® 22IB46 tested was a white to cream coloured powder with a purity of 99.7% and not completely soluble in test medium at the concentrations tested.

 

Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium.

Thereafter, the resulting mixture was filtered and the obtained Saturated Solution (SS) used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.

 

A combined limit/range-finding test was performed, during which, six exponentially growing algal cultures were exposed to an untreated control and the limit concentration, i.e. the undiluted SS. In addition, three replicates per group were exposed to 1.0 and 10% SS in the combined range-finding test. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and after 72 hours of exposure.

 

Samples taken from the highest test concentration were analysed. At the start of the test, the measured concentration was 0.17 mg/L. The concentration did not remain stable during the test duration and was at 49% of initial after 24 hours of exposure and at 11% of initial at the end of the test. Based on these results, the Time Weight Average (TWA) concentration was calculated to be 0.067 mg/L and used to determine the effect parameters.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

The effect parameters, based on the TWA concentration, obtained in this study are summarized in the table below.

 

Parameter

(mg/L)

NOEC

EC10

EC20

EC50

Growth rate

Yield

0.067

0.067

>0.067

<0.067

>0.067

>0.067

>0.067

>0.067

 

It was concluded that the test item had no inhibitory effect on algal growth up to and including a test concentration of 0.067 mg/L, being the TWA concentration in a filtrate prepared at a loading rate of 100 mg/L.

The 72h-NOEC for growth rate inhibition and the 72h-NOEC for yield inhibition were at or above the limit of solubility (0.067 mg/L being the TWA concentration in a filtrate prepared at a loading rate of 100 mg/L).

Description of key information

Key values determined in a GLP accredited laboratory study using Pseudokirchneriella subcapitata, in accordance with OECD Guideline 201 and EU Method C3.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.067 mg/L
EC10 or NOEC for freshwater algae:
0.067 mg/L

Additional information

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with Lowinox® 22IB46.

 

The batch of Lowinox® 22IB46 tested was a white to cream coloured powder with a purity of 99.7% and not completely soluble in test medium at the concentrations tested.

 

Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium.

Thereafter, the resulting mixture was filtered and the obtained Saturated Solution (SS) used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.

 

A combined limit/range-finding test was performed, during which, six exponentially growing algal cultures were exposed to an untreated control and the limit concentration, i.e. the undiluted SS. In addition, three replicates per group were exposed to 1.0 and 10% SS in the combined range-finding test. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and after 72 hours of exposure.

 

Samples taken from the highest test concentration were analysed. At the start of the test, the measured concentration was 0.17 mg/L. The concentration did not remain stable during the test duration and was at 49% of initial after 24 hours of exposure and at 11% of initial at the end of the test. Based on these results, the Time Weight Average (TWA) concentration was calculated to be 0.067 mg/L and used to determine the effect parameters.

 

It was concluded that the test item had no inhibitory effect on algal growth up to and including a test concentration of 0.067 mg/L, being the TWA concentration in a filtrate prepared at a loading rate of 100 mg/L.

The 72h-NOEC for growth rate inhibition and the 72h-NOEC for yield inhibition were at or above the limit of solubility (0.067 mg/L being the TWA concentration in a filtrate prepared at a loading rate of 100 mg/L).

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