Bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-06 to 2017-11-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper

EC Number:

300-491-4

EC Name:

Bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper

Cas Number:

93940-93-3

Molecular formula:

C10H20CuN2O6

IUPAC Name:

bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.800, 0.250, 0.0781, 0.0244, 0.00763 mg/L and control
- Sampling method: taken in the main test at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged solutions from all test item concentrations and control. For each sampling also a retain sample was taken.
- Sample storage conditions before analysis: mixed with 20 µL formic acid and stored deep-frozen (≤ - 18 °C)

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solution: weight necessary amount of test item in volumetric flask and added test medium up to the benchmark, then homogenised by shaking and one minute of sonication. Lower test solutions: dilution appropriate solution with test medium
- Test concentration: 0.800, 0.260, 0.0808, 0.0233, 0.00736 mg/L and control

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany.
- Method of cultivation: grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution.
- Culture conditions: Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 - 120 µEm-2s-1); Temperature: 21 - 24 °C; Culture flasks: 100 mL Erlenmeyer flasks; CO2 supply by shaking on a rotating shaker, approximately 105 rpm

ACCLIMATION
- Acclimation period: 3 to 4 day
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23.0 – 23.3 °C

pH:

7.55 to 7.76 at 0 h and 7.67 to 7.88 at 72 h

Nominal and measured concentrations:

Nominal concentrations: 0.800, 0.250, 0.0781, 0.0244, 0.00763 mg/L
Measured initial concentration: 0.800, 0.260, 0.0808, 0.0233, 0.00736 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps
- Type : closed
- Material, size, headspace, fill volume: volume: 100 mL, fill volume: ~ 50 mL
- Initial cells density: 0.5E+04 cells/mL
- CO2 supplied: by continuous agitation
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, modified AAP-Medium. As the test item contains metal components, the content of Na2EDTA·2H2O in the employed AAP-medium was reduced to 0.100 mg/L (instead of 0.300 mg/L) as recommended by OCSPP 850.4500.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuously from the side
- Light intensity and quality: 75.9 µEm-2s-1 (mean)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement at 24, 48 and 72 hours
- Other: morphological appearance: at the end of the test; pH-value: at t = 0 h and t = 72 h; temperature: measured continuously and recorded at hours 0, 24, 48 and 72 h; light intensity: measured once a year, and confirmed for one representative position at test start; test solution appearance: conducted daily

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Range finding study
- Test concentrations: 100, 10.0, 1.00, 0.100, 0.0100 mg/L and control
- Results used to determine the conditions for the definitive study: After 72 h at termination of the test a clear dose response relation was observed for the inhibition of growth rate and yield from nominal test item concentrations of 0.01 to 1.00 mg/L. The inhibition of growth rate peaked in 126.7 % and the inhibition of yield peaked in 101.4 % at a nominal test item concentration of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, also tested in modified medium

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.07 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.015 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.007 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.069 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.016 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.008 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Morphology evaluation: normal for the control and up to and including a nominal test item concentration of 0.0781 mg/L. No cells were observed at 0.250 and 0.800 mg/L nominal test item concentration.
- Physicochemical Data: pH and temperature during the test, light intensity were within the range specified by OECD 201.
- Chemical analysis of test item concentrations: measured initial concentrations: ranged from 93 % to 104 % of nominal; aged samples: between 98 % and 106 % of nominal.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: growth rate 1.23 mg/L, yield 0.685 mg/L, biomass intergral 0.639 mg/L.
Results were considered valid.

Reported statistics and error estimates:

EC50: Probit analysis following Gompertz distribution for growth rate and following normal distribution for yield
NOEC: Following Dunnetts-t-test (left-sided, p<0.05) for growth rate and for yield

Any other information on results incl. tables

Non-GLP range-finding test:

Percentage inhibition of growth rate and yield observed in the non-GLP range-finding test:

Conc.  [mg/L]	% Inhibition at test end (72 h)
	Growth rate	Yield
Control	0	0
0.0100	3.1	12.0
0.100	32.8	72.2
1.00	119.3	101.2
10.0	119.4	101.2
100	126.7	101.4

Main study - Biological results:

Individual cell numbers：

Concentration (mg/L)	Cell numbers (10^4/mL)				Yield
	0h	24h	48h	72h	0h-72h
Control	0.65	1.76	6.99	27.2	26.55
0.00763		1.76	5.99	22.01	21.36
0.0244		1.28	2.75	12.48	11.83
0.0781		0.8	1.61	5.3	4.65
0.25		0.5	0.33	0.49	-0.16
0.8		0.36	0.15	0.15	-0.5

Percentage inhibition of growth rate:

Conc.	% Inhibition of growth rate
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.00763	-1.2	5.7	5.4
0.0244	32.4	39.0	22.9 *
0.0781	80.4	62.0	43.9 *
0.250	127.8	132.2	108.7 *^1)
0.800	160.2	163.1	141.8 *^2)

¹⁾Value was set to 100 for EC_{10, 20, 50}-calculation

²⁾Value was omitted for EC_{10, 20, 50}-calculation

* Statistically significant different to the control

Percentage inhibition of yield:

Conc.	% Inhibition of yield
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.00763	0.0	15.8	19.5
0.0244	43.2	66.9	55.4 *
0.0781	86.5	84.9	82.5 *
0.250	113.5	105.0	100.6 *^1)
0.800	126.1	107.9	101.9 *^2)

¹⁾Value was set to 100 for EC_{10, 20, 50}-calculation

²⁾Value was omitted for EC_{10, 20, 50}-calculation

* Statistically significant different to the control

Analytical results:

Determined concentration of test item:

Test item nominal concentration [mg/L]	Sampling	Test item measured concentration [mg/L]	% of nominal	Geometric mean [%]	Geometric mean concentration [mg/L]
Control	0 h fresh	< LOD	-	-	-
	72 h aged	< LOD	-
0.00763	0 h fresh	0.00724	95	96.5	0.00736
	72 h aged	0.00750	98
0.0244	0 h fresh	0.0226	93	95.5	0.0233
	72 h aged	0.0240	98
0.0781	0 h fresh	0.0810	104	103.5	0.0808
	72 h aged	0.0805	103
0.250	0 h fresh	0.256	102	104.0	0.260
	72 h aged	0.266	106
0.800	0 h fresh	0.810	101	100.0	0.800
	72 h aged	0.790	99

Effect values:

NOEC, LOEC, ECx-values and 95 % Confidence Intervals (0 - 72 hours):

	Test item[mg/L]
	nominal	measured (geometric mean)
ErC10(Growth rate)	0.0159	0.0153
95 % confidential limits	1.10 · 10^-9– 0.0423	7.36 · 10^-19– 0.0437
ErC20	0.0284	0.0280
95 % confidential limits	0.000000613 – 0.0641	7.25 · 10^-13– 0.0672
ErC50	0.0685	0.0695
95 % confidential limits	0.00408 – 0.254	0.000296 – 0.356
EyC10(Yield)	0.00482	0.00450
95 % confidential limits	0.00319 – 0.00654	0.00295 – 0.00616
EyC20	0.00805	0.00763
95 % confidential limits	0.00584 – 0.0103	0.00549 – 0.00982
EyC50	0.0215	0.0210
95 % confidential limits	0.0177 – 0.0257	0.0172 – 0.0252
NOEC	0.00763	0.00736
LOEC	0.0244	0.0233

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibitory effects were determined for growth rate and for yield at test item concentrations of 0.0244 mg/L (nominal) and 0.0233 mg/L (measured) and above. The EC10 for growth rate was set at 0.0153 mg/L (measured) and for yield at 0.0045 mg/L (measured). The EC50-value for growth rate (ErC50) was determined to be 0.0695 mg/L (measured). The EC50-value for yield (EyC50) was 0.0210 mg/L (measured).

Executive summary:

The effects of test item on the growth of the single cell green alga Pseudokirchneriella subcapitata were determined according to OECD 201.

Following a static non-GLP range-finding test with nominal test item concentrations of 100, 10.0, 1.00, 0.100, 0.0100 mg/L and control, a static main test with nominal test item concentrations of 0.800, 0.250, 0.0781, 0.0244 and 0.00763 mg/L and control was performed.

Six replicates were employed for the control and three for each test item concentration. The test was performed in 100 mL Erlenmeyer flasks each containing ~ 50 mL test solution. The pH was recorded at test start and test end. Temperature was measured continuously over the whole test period and recorded daily. Light intensity of the continuous illumination was measured at test start.

All validity criteria met for the study.

Test concentrations were analytically verified using HPLC-MS/MS. The measured initial concentrations of test item in the samples taken at test start were within 80 – 120 %.

Significant inhibitory effects were determined for growth rate and for yield at test item concentrations of 0.0244 mg/L (nominal) and 0.0233 mg/L (measured) and above. The EC10 for growth rate was set at 0.0153 mg/L (measured) and 0.0045 mg/L (measured) for yield. The EC50-value for growth rate (ErC50) was determined to be 0.0685 mg/L (nominal) and 0.0695 mg/L (measured). The EC50-value for yield (EyC50) was 0.0215 mg/L (nominal) and 0.0210 mg/L (measured).