Trisodium 2-(carboxylatomethyl(2-hydroxyethyl)amino)ethyliminodi(acetate)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

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 for cross-reference:

read-across source

Qualifier:

according to guideline

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. QA statement)

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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.

Reference 2

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 for cross-reference:

read-across source

Qualifier:

according to guideline

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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.

Reference 3

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 for cross-reference:

read-across source

Qualifier:

according to guideline

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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.

Reference 4

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 for cross-reference:

read-across source

Qualifier:

according to guideline

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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.

Reference 5

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 for cross-reference:

read-across source

Qualifier:

according to guideline

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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.

Reference 6

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

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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.

Reference 7

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 for cross-reference:

read-across source

Qualifier:

according to guideline

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)

Reference 8

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 for cross-reference:

read-across source

Qualifier:

according to guideline

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).

Reference 9

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.

Reference 10

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

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

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.

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.