Dipotassium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cuprate(2-)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22-9-2009 - 25-9-2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted to OECD guidelines, was compliant to GLP, analysis certificate is present, chemical analysis was conducted and validility criteria were met.

Reason / purpose for cross-reference:

reference to other study

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:

EC10

Effect conc.:

42.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.000109 - 155.94

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

649.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 204.4 - 18430

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

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.

Description of key information

Primarily studies with equimolar EDTA vs essential nutrients shall be considered to conclude on toxicity rather than that depletion inhibits growth. The studies of BASF & AkzoNobel with the EDTA-Fe complex can be considered appropriate as well as the study by Geurts (2009). Geurts (2009) study is the preferred (key) information based on measured concentrations and the most conservative ErC10/ErC50 for EDTA-MnNa2 value of 39.5/699.3 mg a.i./L. When recalculated to EDTA-CuK2 using the MW ratio the ErC10/ErC50 would be 38.3/678.4 mg/L (*429.95/443.17).

Key value for chemical safety assessment

EC50 for freshwater algae:

678.4 mg/L

EC10 or NOEC for freshwater algae:

38.3 mg/L

Additional information

Multiple algae tests (Scenedesmus subspicatus, Scenedesmus quadricauda, Pseudokirchneriella subcapitata) with different EDTA salts and complexes have been performed. As chelating agent, available EDTA will interact with essential micro and macro nutrients to the growth of algae. If a surplus of EDTA is present, it will cause depletion of nutrients thus growth inhibition, as shown in the results from Dufkova (1984), BASF (1995, 2x), AkzoNobel (2001), Kean (2009 & 2009 1-5) and Geurts (2009).