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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
22-9-2009 - 25-9-2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read across study
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
See Below
Principles of method if other than guideline:
The NaHCO3 concentration of the test medium was 150 mg/L instead of 50 mg/L, in order to help maintain a more constant pH during the test.
The Fe-EDTA and trace elements components of the OECD media were increased to 150% of the standard composition, in order to prevent
nutrient deprivation during the test.
Increase in pH was greater than 1.5 units in the one of the control and lowest concentration replicates.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
At the start of the test, samples of 2.0 mL were taken per replicate from test solutions containing a nominal concentration of 0, 15, 154 and
1573 mg/L just before the addition of the algae at T0. For the samples at the end of the test, 2 mL of the contents of each test vessel mentioned above was sampled and filtered using a GP 0.45 µm. Samples at T0 were stored for 24 hours until analysis in the refrigerator and T72 samples were
analyzed immediately.
Vehicle:
no
Details on test solutions:
The following final nominal test concentrations were prepared: 0, 15.0, 48.0, 154.0, 492.0 and 1573.0 mg/L
The test solutions were prepared by addition of the adequate amounts of stock solution to the test vessels, then dilution water was added up to the
required final volume. A control containing only test medium was included in the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Freshwater unicellular algae P. subcapitata (CCAP 278/4) obtained from the Culture Collection of Algae and Protozoa, Dunstaffnage Marine
Laboratory, Oban, Argyll, Scotland, UK.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
At the end of the test five random samples were microscopically checked for purity of the algal culture. No contamination was observed.
Hardness:
-
Test temperature:
The temperature varied from 22.35 to 22.9 °C during the test.
pH:
Nominal test
concentration
(mg/L) Time(hours)
0 72
0 I(Control) 8.0 9.9
0 II 8.0 9.9
0 III 8.0 9.9
0 IV 8.0 9.9
0 V 8.0 10.0
0 VI 8.0 9.9
15 I 8.0 9.9
15 II 8.0 9.8
15 III 8.0 10.0
48 I 8.0 9.6
48 II 8.0 9.5
48 III 8.0 9.5
154 I 8.0 8.5
154 II 8.0 8.5
154 III 8.0 8.5
492 I 8.1 8.4
492 II 8.1 8.4
492 III 8.1 8.4
1573 I 8.1 8.4
1573 II 8.1 8.4
1573 III 8.1 8.4

Dissolved oxygen:
-
Salinity:
-
Nominal and measured concentrations:
Sample Nominal conc. (mg/L) Calculated conc. (mg/L) Recovery (%)
Control, t=0 0 15 mg/L, t=0 15 12.6 84.0
154 mg/L, t=0 (*) 154 128.3 83.3
1573 mg/L, t=0 (**) 1573 1298.9 82.6
control ST 30 mg/L 30.60* 30.8 100.5
(*): Sample was diluted 5 times in order to be able to determine the concentration
(**): Sample was diluted 50 times in order to be able to determine the concentration
Details on test conditions:
The test was carried out in a temperature controlled illuminated orbital incubator, in which the temperature was maintained at 23 ± 2°C, and a
continuous uniform illumination was provided in the spectral range of 400 to 700 nm by using fluorescent lamps at a distance of about
0.36 +/- 0.02 m from the algal cultures. The light intensity was in the range of 60 to 120 µE•m-2•s-1 (= µmol.m-2•s-1). The test vessels were
swirled continuously at a speed sufficient to prevent sedimentation of the algae.
Reference substance (positive control):
yes
Remarks:
As part of GLP maintenance
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
649.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Remarks on result:
other: 204.4 - 18430
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
42.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Remarks on result:
other: 0.000109 - 155.94
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Details on results:
The results reported are based on nominal concentrations.
The test substance did cause inhibition of growth rate and therefore ErC10 and ErC50 values could be calculated. The ErC10 (72h) was calculated as 42.8 mg/L. The ErC50 (72h) was calculated as 649.3 mg/L. All values are reported for MnNa2-EDTA as received.
Results with reference substance (positive control):
As part of annual maintenance a reference test was performed to check the sensitivity of the alga used in this study.
The EC50 values of the reference compound, potassium dichromate, were 1.27 mg/l with 95% fiducial limits of 0.85 - 1.74 mg/l based on
growth rate and 0.71 mg/l with 95% fiducial limits 0.48 - 1.06 mg/l of based on biomass. (Documented as part of GLP laboratory maintenance)
The EC50 values of the reference compound, potassium dichromate, should be in the range of 0.25-2.0 mg/L Therefore, the recommended criteria were fulfilled.
Reported statistics and error estimates:
The Lowest Observed Effect Concentration (LOEC) was determined by comparison of the growth at each concentration and the control using
threshold values from the William’s test.
The No Observed Effect Concentration (NOEC) was derived from the results as the first concentration below the LOEC value, where growth shows no
significant inhibition relative to the control values.
Confidence limits were computed on the basis of Fieller’s theorem if possible. All computations were performed using the
TOXCALC TM version 5.0 program.

 Absorbance measured in the algal growth inhibition test with MnNa2-EDTA

 

Concentration

Time (hours)

 

Area

Inhibition

Specific

Inhibition

(mg/L)

0

24

48

72

 

   (%)

Growth rate

   (%)

0

0.006

0.035

0.231

0.919

17.052

 

0.070

 

0

0.005

0.034

0.22

0.911

16.728

 

0.072

 

0

0.006

0.034

0.208

0.881

16.02

 

0.069

 

0

0.005

0.034

0.202

0.889

16.032

 

0.072

 

0

0.005

0.035

0.218

0.913

16.728

 

0.072

 

0

0.005

0.036

0.212

0.883

16.248

 

0.072

 

Mean

0.005

0.034

0.215

0.899

16.468

 

0.071

0

 

 

 

 

 

 

 

 

 

15

0.006

0.035

0.223

0.881

16.404

 

0.070

 

15

0.005

0.034

0.188

0.802

14.652

 

0.070

 

15

0.005

0.033

0.214

0.868

16.044

 

0.072

 

Mean

0.005

0.034

0.208

0.850

15.7

4.663

0.070

1.051

 

 

 

 

 

 

 

 

 

48

0.006

0.035

0.179

0.798

14.352

 

0.067

 

48

0.005

0.035

0.176

0.746

13.716

 

0.069

 

48

0.005

0.033

0.169

0.755

13.608

 

0.069

 

Mean

0.005

0.034

0.174

0.76

13.892

15.642

0.068

3.945

 

 

 

 

 

 

 

 

 

154

0.005

0.031

0.082

0.191

4.704

 

0.048

 

154

0.005

0.031

0.085

0.203

4.92

 

0.049

 

154

0.005

0.032

0.095

0.233

5.544

 

0.051

 

Mean

0.005

0.031

0.0873

0.209

5.056

69.298

0.050

30.104

 

 

 

 

 

 

 

 

 

492

0.006

0.027

0.046

0.098

2.568

 

0.038

 

492

0.006

0.025

0.041

0.084

2.232

 

0.036

 

492

0.006

0.027

0.042

0.081

2.268

 

0.035

 

Mean

0.006

0.026

0.043

0.087

2.356

85.693

0.036

48.420

 

 

 

 

 

 

 

 

 

1573

0.007

0.024

0.031

0.045

1.44

 

0.027

 

1573

0.007

0.023

0.031

0.045

1.416

 

0.027

 

1573

0.007

0.023

0.031

0.045

1.416

 

0.027

 

Mean

0.007

0.023

0.031

0.045

1.424

91.352

0.027

61.718

 

 

Validity criteria fulfilled:
yes
Conclusions:
This study can be concluded reliable without restrictions.
Executive summary:

This study was conducted to OECD guideline 201 and was compliant to GLP. An analysis certificate was present and chemical analysis was conducted. Validility criteria was met. All deviations from guideline and study plan were clearly reported and justified. Preliminary testing was also conducted. See reference below.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
15-9-2009 - 18-9-2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
The NaHCO3 concentration of the test medium was 150 mg/l in stead of 50 mg/l, in order to maintain a more constant pH during the test.
In this test the concentration of the trace elements and the Fe-EDTA components of the OECD test medium were enriched up to 150%.
No chemical analysis was conducted, due to this test being a non-GLP screening test.
GLP compliance:
no
Remarks:
GLP accredited laboratory not conducted to GLP
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
886.89 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
84.75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

Test Chemical MnNa2-EDTA additional testing raw data (150% enriched)

 

Concentration

Time (hours)

Area

Inhibition

Specific

Inhibition

(mg/L)

0

72

 

 

Percentage

Growth Rate

Percentage

0

0.007

0.71

 

25.308

 

0.064157706

 

0

0.006

0.717

 

25.596

 

0.06643495

 

0

0.006

0.705

 

25.164

 

0.066200532

 

0

0.006

0.803

 

28.692

 

0.068008267

 

Mean

0.00625

0.73375

 

26.19

 

0.066188707

0

 

 

 

 

 

 

 

 

1

0.006

0.675

 

24.084

 

0.065034743

 

1

0.006

0.798

 

28.512

 

0.067358894

 

Mean

0.006

0.7365

 

26.298

-0.4123711

0.066245377

-0.0856191

 

 

 

 

 

 

 

 

10

0.006

0.832

 

29.736

 

0.067938214

 

10

0.006

0.802

 

28.656

 

0.067428317

 

Mean

0.006

0.817

 

29.196

-11.477663

0.067685605

-2.2615614

 

 

 

 

 

 

 

 

100

0.007

0.461

 

16.344

 

0.058657126

 

100

0.006

0.361

 

12.78

 

0.057485908

 

Mean

0.0065

0.411

 

14.562

44.3986254

0.058130601

12.174442

 

 

 

 

 

 

 

 

1000

0.01

0.065

 

1.98

 

0.031699755

 

1000

0.01

0.063

 

1.908

 

0.031238671

 

Mean

0.01

0.064

 

1.944

92.5773196

0.031471127

52.4524229

Validity criteria fulfilled:
yes
Conclusions:
This study can be concluded reliable with restrictions.
Executive summary:

The study concerned was in accordance with OECD guidelines in a GLP accredited laboratory, but was not GLP audited. No chemical analysis was performed. Certificate of analysis was available (see attachment). Control growth was satisfactory and current reference test with potassium dichromate had been conducted on the algae species used for testing. This study can be considered reliable with the restrictions mentioned. And is therfore reliable as supporting evidence.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
15-9-2009 - 18-9-2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
The NaHCO3 concentration of the test medium was 150 mg/l in stead of 50 mg/l, in order to maintain a more constant pH during the test.
In this test the concentration of the trace elements and the Fe-EDTA components of the OECD test medium were enriched up to 200%.
No chemical analysis was conducted, due to this test being a non-GLP screening test.
GLP compliance:
no
Remarks:
GLP accredited laboratory not conducted to GLP
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1 028.53 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
74.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

Test Chemical MnNa2-EDTA additional testing raw data (200% enriched)

 

Concentration

Time (hours)

Area

Inhibition

Specific

Inhibition

 

 

 

 

 

Percentage

Growth Rate

Percentage

(mg/L)

0

72

 

 

 

 

 

0

0.006

0.808

 

28.872

 

0.06809448

 

0

0.006

0.791

 

28.26

 

0.067799146

 

0

0.006

0.785

 

28.044

 

0.067693392

 

0

0.006

0.741

 

26.46

 

0.066892238

 

Mean

0.006

0.78125

 

27.909

 

0.067626885

0

 

 

 

 

 

 

 

 

1

0.006

0.686

 

24.48

 

0.065821086

 

1

0.006

0.654

 

23.328

 

0.065157609

 

Mean

0.006

0.67

 

23.904

14.3502096

0.065493309

3.15492311

 

 

 

 

 

 

 

 

10

0.006

0.752

 

26.856

 

0.067096901

 

10

0.006

0.838

 

29.952

 

0.068600814

 

Mean

0.006

0.795

 

28.404

-1.7736214

0.067869203

-0.3583164

 

 

 

 

 

 

 

 

100

0.006

0.35

 

12.384

 

0.056474635

 

100

0.006

0.486

 

17.28

 

0.061034016

 

Mean

0.006

0.418

 

14.832

46.855853

0.058940583

12.8444513

 

 

 

 

 

 

 

 

1000

0.012

0.08

 

2.448

 

0.033874958

 

1000

0.011

0.072

 

2.196

 

0.034617415

 

Mean

0.0115

0.076

 

2.322

91.6801032

0.034220184

49.3985509

Validity criteria fulfilled:
yes
Conclusions:
This study can be concluded reliable with restrictions.
Executive summary:

The study concerned was in accordance with OECD guidelines in a GLP accredited laboratory, but was not GLP audited. No chemical analysis was performed. Certificate of analysis was available (see attachment). Control growth was satisfactory and current reference test with potassium dichromate had been conducted on the algae species used for testing. This study can be considered reliable with the restrictions mentioned. And is therfore reliable as supporting evidence.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
22-7-2009 - 25-7-2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
The NaHCO3 concentration of the test medium was 150 mg/l in stead of 50 mg/l, in order to maintain a more constant pH during the test.
GLP compliance:
no
Remarks:
GLP accredited laboratory not conducted to GLP
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1 165.47 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
16.88 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

Test Chemical MnNa2-EDTA

 

 

Concentration

Time (hours)

Area

Inhibition

Specific

Inhibition

(mg/L)

0

72

 

 

percentage

Growth Rate

Percentage

0

0.007

0.501

 

17.784

 

0.059315222

 

0

0.007

0.492

 

17.46

 

0.059063452

 

0

0.006

0.35

 

12.384

 

0.056474635

 

0

0.008

0.285

 

9.972

 

0.049625662

 

0

0.007

0.464

 

16.452

 

0.058249644

 

0

0.008

0.265

 

9.252

 

0.048615115

 

Mean

0.007167

0.392833

 

13.884

 

0.055610344

0

 

 

 

 

 

 

 

 

1

0.006

0.372

 

13.176

 

0.054897007

 

1

0.006

0.46

 

16.344

 

0.057837762

 

 

 

 

 

 

 

 

 

Mean

0.006

0.416

 

14.76

-6.3094209

0.056445072

-1.5010294

 

 

 

 

 

 

 

 

10

0.006

0.333

 

11.772

 

0.053363878

 

10

0.006

0.279

 

9.828

 

0.050915889

 

 

 

 

 

 

 

 

 

Mean

0.006

0.306

 

10.8

22.2126188

0.052193748

6.143814621

 

 

 

 

 

 

 

 

100

0.007

0.116

 

3.924

 

0.038688685

 

100

0.007

0.157

 

5.4

 

0.04288703

 

 

 

 

 

 

 

 

 

Mean

0.007

0.1365

 

4.662

66.4217805

0.040945892

26.37000805

 

 

 

 

 

 

 

 

1000

0.012

0.062

 

1.8

 

0.028840699

 

1000

0.012

0.067

 

1.98

 

0.030005254

 

 

 

 

 

 

 

 

 

Mean

0.012

0.0645

 

1.89

86.3872083

0.029435179

47.06887874

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
This study can be concluded reliable with restrictions.
Executive summary:

The study concerned was in accordance with OECD guidelines in a GLP accredited laboratory, but was not GLP audited. No chemical analysis was performed. Certificate of analysis was available (see attachment). Control growth was satisfactory and current reference test with potassium dichromate had been conducted on the algae species used for testing. This study can be considered reliable with the restrictions mentioned. And is therefore reliable as supporting evidence.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
15-9-2009 - 18-9-2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
The NaHCO3 concentration of the test medium was 150 mg/l in stead of 50 mg/l, in order to maintain a more constant pH during the test.
In this test the concentration of the trace elements and the Fe-EDTA components of the OECD test medium were enriched up to 120%.
No chemical analysis was conducted, due to this test being a non-GLP screening test.
GLP compliance:
no
Remarks:
GLP accredited laboratory not conducted to GLP
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
769.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
31.95 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

Test Chemical MnNa2-EDTA additional testing raw data (120% enriched) 

 

Concentration

Time (hours)

Area

Inhibition

Specific

Inhibition

(mg/L)

0

72

 

 

Percentage

Growth rate

Percentage

0

0.006

0.743

 

26.532

 

0.066929675

 

0

0.005

0.803

 

28.728

 

0.070540511

 

0

0.005

0.818

 

29.268

 

0.070797561

 

0

0.005

0.803

 

28.728

 

0.070540511

 

Mean

0.00525

0.79175

 

28.314

 

0.069666911

0

 

 

 

 

 

 

 

 

1

0.005

0.803

 

28.728

 

0.069867193

 

1

0.005

0.632

 

22.572

 

0.066542456

 

Mean

0.005

0.7175

 

25.65

9.40877305

0.068304073

1.95622053

 

 

 

 

 

 

 

 

10

0.005

0.78

 

27.9

 

0.069463699

 

10

0.005

0.664

 

23.724

 

0.067228202

 

Mean

0.005

0.722

 

25.812

8.83661793

0.068390879

1.83161929

 

 

 

 

 

 

 

 

100

0.005

0.239

 

8.424

 

0.053045569

 

100

0.005

0.301

 

10.656

 

0.056246006

 

Mean

0.005

0.27

 

9.54

66.3064209

0.054737769

21.4293153

 

 

 

 

 

 

 

 

1000

0.01

0.056

 

1.656

 

0.029492478

 

1000

0.008

0.056

 

1.728

 

0.034848901

 

Mean

0.009

0.056

 

1.692

94.0241577

0.031914053

54.1905154

 

Validity criteria fulfilled:
yes
Conclusions:
This study can be concluded reliable with restrictions.
Executive summary:

The study concerned was in accordance with OECD guidelines in a GLP accredited laboratory, but was not GLP audited. No chemical analysis was performed. Certificate of analysis was available (see attachment). Control growth was satisfactory and current reference test with potassium dichromate had been conducted on the algae species used for testing. This study can be considered reliable with the restrictions mentioned. And is therfore reliable as supporting evidence.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
15-9-2009 - 18-9-2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
The NaHCO3 concentration of the test medium was 150 mg/l in stead of 50 mg/l, in order to maintain a more constant pH during the test.
GLP compliance:
no
Remarks:
GLP accredited laboratory not conducted to GLP
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
652.66 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
11.39 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
MnNa2-EDTA
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

Test Chemical MnNa2-EDTA additional testing raw data (standard media)

  

Concentration

Time (hours)

Area

Inhibition

Specific Growth

Inhibition

(mg/L)

0

72

 

 

Percentage

Rate

Percentage

0

0.005

0.755

 

27

 

0.069684442

 

0

0.006

0.924

 

33.048

 

0.069957675

 

0

0.006

0.879

 

31.428

 

0.069264242

 

0

0.006

0.813

 

29.052

 

0.068180162

 

Mean

0.00575

0.84275

 

30.132

 

0.069270424

0

 

 

 

 

 

 

 

 

1

0.006

0.727

 

25.956

 

0.067213648

 

1

0.006

0.818

 

29.232

 

0.068852178

 

Mean

0.006

0.7725

 

27.594

8.42293907

0.068057062

1.75162986

 

 

 

 

 

 

 

 

10

0.006

0.743

 

26.532

 

0.067516106

 

10

0.006

0.572

 

20.376

 

0.063881972

 

Mean

0.006

0.6575

 

23.454

22.1624851

0.065817564

4.98460874

 

 

 

 

 

 

 

 

100

0.006

0.179

 

6.228

 

0.047733171

 

100

0.006

0.166

 

5.76

 

0.046684455

 

Mean

0.006

0.1725

 

5.994

80.1075269

0.047218709

31.8342423

 

 

 

 

 

 

 

 

1000

0.01

0.063

 

1.908

 

0.032279046

 

1000

0.01

0.062

 

1.872

 

0.032040604

 

Mean

0.01

0.0625

 

1.89

93.7275986

0.032160337

53.5727733

Validity criteria fulfilled:
yes
Conclusions:
This study can be concluded reliable with restrictions.
Executive summary:

The study concerned was in accordance with OECD guidelines in a GLP accredited laboratory, but was not GLP audited. No chemical analysis was performed. Certificate of analysis was available (see attachment). Control growth was satisfactory and current reference test with potassium dichromate had been conducted on the algae species used for testing. This study can be considered reliable with the restrictions mentioned. And is therfore reliable as supporting evidence.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: EEC guideline 79/831/EEC, Annexe V, part C: Methods for the determination of ecotoxicity, algae: Test of growth inhibition, May 1988
GLP compliance:
yes
Analytical monitoring:
yes
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Test species: Scenedesmus subspicatus
Strain: CHODAT SAG 86.81
Source: Algae collection Goettingen, SAG 86.81
Test type:
static
Water media type:
freshwater
Limit test:
no
Test temperature:
23 +/- 2 °C
pH:
7.65 - 8.80
Details on test conditions:
Aeration: none
No. of replicates:
3 inoculated replicates
1 uninoculated replicate for measuring the pH-value of the uninoculated test concentrations after an incubation time of 72 h.
Controls: negative control

TEST MEDIUM / WATER PARAMETERS
Dilution water: according to EEC guideline 79/831/EEC, Annexe V, part C:
Methods for the determination of ecotoxicity, algae: Test of growth inhibition, May 1988 and the SOP of the laboratory
Deviation from the SOP: Each test concentration was mixed according to the following regimen:
1 part of inoculum (1E+05 cells/ml in test medium)
1 part of test medium (10 times concentrated)
8 parts of test substance (in appropriate water dilutions)

TEST CONDITIONS
Illumination: 120 uEinstein/m*m*s (permanent Illumination, Color of light was universal white, L 25 (Osram))
Inoculation density: 10000 cells/mL (initial concentration in the test)

TEST CONCENTRATIONS
Test concentrations: 100 mg/l - 0.39 mg/L
Stock solution: 125 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.77 mg/L
Conc. based on:
test mat.
Remarks:
Na4-EDTA
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.7 mg/L
Conc. based on:
test mat.
Remarks:
Na4-EDTA
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.39 mg/L
Conc. based on:
test mat.
Remarks:
Na4-EDTA
Basis for effect:
growth rate
Effect on the development of biomass [mg/L]:
EbC 10 = 0.48 mg/L
EbC 50 = 1.01 mg/L
EbC 90 = 100 mg/L

Effect on growth rate [mg/L]:
ErC 10 = 0.70 mg/L
ErC 50 = 2.77 mg/L
ErC 90 => 100.0 mg/L

No observed effect concentration (95 % significance level):
NOEC (72 h) = 0.39 mg/L

Lowest observed effect concentration:
LOEC (72 h) = 0.78 mg/L

VALIDTY CRITERIA:
The validity criteria were maintained.
Factor of cell division (cell count) after 72 hours: 46

SUMMARY OF THE CONCENTRATION CONTROL ANALYSIS:
The concentrations 100 and 12.5 mg/L, the untreated control and the stock solution (125 mg/L) were analysed. Test
conditions and results are listed in the GLP-reports 94LOO650 and 94LOO663, dated 22 Nov 1994, from Dr. Scholtissek, BASF AG, ZAX/OF.

Time scan:
begin of test (0 h) - uninoculated replicate
end of test (72 h) - uninoculated replicate
end of test (72 h) - inoculated replicate after removal of algal cells through centrifugation

Recovery in %:
control / test / stock solution /
-----------------------------------------------------------
t= 0 h uninoculated <DL / 100-104 / 99.2 /
t= 72 h uninoculated <DL / 96-99 / 98.4 /
t= 72 h inoculated <DL / 99-104 / - /
-----------------------------------------------------------
DL= detection limit (1 mg/L)
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study with the read across substance is considered sufficient to fulfil the information requirements.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
modified test system without iron limitation (medium differing to OECD)
Principles of method if other than guideline:
The study was conducted according to the EU guideline EEC/79/831, Annex V, C, Algae and the standard operating procedures (SOP) of the BASF laboratory.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Stock solutions:
249.75 mg/L (Na4-EDTA)
177.55 mg/L (FeCl3 x 6 H20)
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Test species: Scenedesmus subspicatus
Strain: CHODAT SAG 86.81
Source: Algae collection Goettingen, SAG 86.81
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 +/- 2 °C
pH:
ca. 8
Nominal and measured concentrations:
Test concentrations: 263.04 µmol/L - 1.03 µmol/L
Details on test conditions:
TEST CONDITIONS
Illumination: 120 µEinstein/m*m*s (permanent Illumination, Color of light was universal white, L 25 (Osram))
Inoculation density: 10000 cells/mL (initial concentration in the test)

TEST SYSTEM
Aeration: none
No. of replicates:
3 inoculated replicates
1 uninoculated replicate for measuring the pH-value of the uninoculated test concentrations after an incubation time of 72 h.
Controls: negative control

TEST MEDIUM / WATER PARAMETERS
Dilution water: OECD medium differing to the SOP of the laboratory
Each test concentration was mixed according to the following regimen:
1 part algal cell suspension in OECD medium
1 part OECD medium 9-times concentrated
4 parts FeCl3 x 6 H20 dilutions in H20
4 parts Na4-EDTA dilutions in H20
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The validity criteria were fulfilled.
Factor of the cell proliferation (cell count) after 72 h: 36.7
Effect on Biomass:
------------------
EbC 10 = > 263.04 umol/l (> 100 mg/l Na4-EDTA)
EbC 50 = > 263.04 umol/l (> 100 mg/l Na4-EDTA)
EbC 90 = > 263.04 umol/l (> 100 mg/l Na4-EDTA)

Effect on Growth rate:
----------------------
ErC 10 = > 263.04 umol/l (> 100 mg/l Na4-EDTA)
ErC 50 = > 263.04 umol/l (> 100 mg/l Na4-EDTA)
ErC 90 = > 263.04 umol/l (> 100 mg/l Na4-EDTA)

NOEC (72 h) = 263.04 umol/l (100 mg/l Na4-EDTA)

LOEC (72 h) = > 263.04 umol/l (> 100 mg/l Na4-EDTA)


Observations:
-------------
Over the whole range of test concentrations, a stimulation of the cell proliferation was observed (max. 30.4 % based on biomass).
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Principles of method if other than guideline:
Not reported
GLP compliance:
not specified
Analytical monitoring:
not specified
Details on sampling:
Not reported
Vehicle:
not specified
Details on test solutions:
Not reported
Test organisms (species):
other: Not reported
Details on test organisms:
Not reported
Test type:
not specified
Water media type:
not specified
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
Not reported
Test temperature:
Not reported
pH:
Not reported
Dissolved oxygen:
Not reported
Salinity:
Not reported
Nominal and measured concentrations:
Not reported
Details on test conditions:
Not reported
Reference substance (positive control):
not specified
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
26 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Remarks on result:
other: No information on medium
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
20 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Duration:
72 h
Dose descriptor:
EC90
Effect conc.:
> 500 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
not specified
Details on results:
Not reported
Results with reference substance (positive control):
Not reported
Reported statistics and error estimates:
Not reported

Not reported

Conclusions:
For a 72-hr test with algae, the EC20 was 20 mg/L, the EC50 was 26 mg/L, and EC90 was >500 mg/L of test substance.
Executive summary:

For a 72-hr test with algae, the EC20 was 20 mg/L, the EC50 was 26 mg/L, and EC90 was >500 mg/L of test substance.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1 to 4 November 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: a nominal 100 mg/L solution was prepared in dilution water, shaken vigorously and allowed to stand for one hour. Test concentrations were prepared by mixing specified quantities of the sample with culture medium to obtain the required concentrations.
- Evidence of undissolved material: a clear colorless solution was obtained
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Common name: marine algae
- Strain: 1077/5
- Source: Culture collection of algae and protozoa, Dunstaffnage Marine Laboratory, Scotland
- Age of inoculum: Not reported
- Method of cultivation: temperature = 20 degrees C, illumination 6000-10000 lux continuous white light, shaking at 200 rpm

ACCLIMATION: Not reported
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not reported
Hardness:
Not reported
Test temperature:
20 +/- 1 degree C
pH:
7.9 - 8.1 (See Table 1)
Dissolved oxygen:
Not reported
Salinity:
Not reported
Nominal and measured concentrations:
Nominal (mg/L): 0, 5.6, 10, 18, 32, 56
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical flask
- Material, size, headspace, fill volume: filled with 200 mL of media
- Aeration: Not applicable
- Initial cells density: 10000 cells/ml
- Control end cells density: 737800 cells/ml
- No. of vessels per concentration (replicates): 3 replicates per concentration
- No. of vessels per control (replicates): 6 control flasks
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: seawater collected on 11 October 1999 from the CEFAS laboratories at Burnham-on-Crouch directly from the River Crouch estuary.
- See Table 2 for composition of Culture Medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not reported
- Photoperiod: continuous white light
- Light intensity and quality: 6000 - 10000 lux
- Salinity (for marine algae): not reported

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: haemocytometer and microscope

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 2x
- Range finding study
- Test concentrations: not reported
- Results used to determine the conditions for the definitive study: 72 hr EC50 approximately 10 mg/L
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
12.06 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Versenol 120/chel)
Basis for effect:
growth rate
Remarks on result:
other: Standard medium
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.9 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Na3-HEDTA
Basis for effect:
growth rate
Remarks on result:
other: Standard medium
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
at 41%
Basis for effect:
biomass
Details on results:
Not reported
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Growth curves for each test concentration were plotted as logarithm of the mean cell density against time. The inhibition of growth was calculated using both the biomass integral (area under the growth curve) and the growth rate method. The EC50 values were estimated where possible using a log-linear or log-probit plot of concentration and percent growth inhibition.

Table 3. Percent inhibition by biomass integral and growth rate.

 Nominal Conc. (mg/L)  Percent inhibition by biomass integral        Percent inhibition by growth rate
   48 hours  72 hours  48 hours  72 hours
 5.6  30  21  4  5
 10  73  74  30  34
 18  99  99  100  84
 32  100  100  100  100
 56  100  100  100  100
Validity criteria fulfilled:
yes
Conclusions:
The 72 hours EC50 of Versenol/120 Chel to Skeleonema costatum was estimates as 3.3 mg/L using the biomass integral and 4.9 mg/L by growth rate calculation.
Executive summary:

The 72 hours EC50 of Versenol/120 Chel to Skeleonema costatum was estimates as 3.3 mg/L using the biomass integral and 4.9 mg/L by growth rate calculation.

Description of key information

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

A number of studies have investigated the toxicity to freshwater or marine algae of trisodium HEDTA and the structural analogues tetrasodium EDTA and magnesium disodium EDTA. The 72 h ErC50 values ranged from 2.77 mg Na4-EDTA/L in standard medium (Scenedesmus (Desmodesmus) subspicatus) to >>100 mg/L in iron-complemented and pH-buffered medium (Pseudokirchneriella subcapitata). The EC10 values from studies using MnNa2-ETDA in supplemented medium ranged from 10 to 74.8 mg MnNa2-EDTA/L (Pseudokirchneriella subcapitata).

As discussed in Appendix I of the read across justification (see Section 13 of the IUCLID file), HEDTA and it structural analogue EDTA are chelators which complex preferentially metals. A series of studies conducted with tetrasodium EDTA showed that its marked toxicity to Scenedesmus subspicatus resulted from the complexation of essential ferrous ions in the test medium. This was also evidenced in testing with MnNa2-ETDA in supplemented medium, where the 72 h ErC50s were all above 100 mg/L.

Overall, Na3-HEDTA is therefore not considered to be directly toxic to algae, so that EC50 values of 100 mg/L are retained for the CSA. A study in a marine species with a 72 h ErC50 of 4.9 mg Na3-HEDTA/L exists but this was not used for the CSA as the study used standard medium, so that toxicity was probably due also here to iron complexation.