Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

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:

2017-12-11 to 2018-03-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

EC Number:

284-943-5

EC Name:

Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

Cas Number:

84989-53-7

Molecular formula:

C42H44CoN12.Cl

IUPAC Name:

hydroxylamine

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 4.00, 1.25, 0.391, 0.122, 0.0381, 0.0119 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: all samples were analysed immediately after sampling

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 20 minutes of ultrasonication. A clear, transparent and turquoise solution was obtained. For lower test solutions: dilution appropriate solution with test medium.
- Test concentration: 4.00, 1.25, 0.391, 0.122, 0.0381, 0.0119 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:

22.6 – 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: 4.00, 1.25, 0.391, 0.122, 0.0381, 0.0119 mg/L and 0 mg/L
Measured concentrations (geometric mean): 0.492, 0.111, 0.0708, 0.0555, 0.0339, < LOQ and 0 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: 88.3 µEm-2s-1 (mean)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorescence measurement at 24, 48 and 72 hours. Since the test item fluoresced by itself, a correction was done using a blank measurement, i.e. solution without algae.
- 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: 10.0, 5.00, 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 concentration response relation was observed for the inhibition of growth rate and yield from nominal test item concentrations of 0.0100 to 10.0 mg/L. The inhibition of growth rate peaked at 160.1 % and the inhibition of yield peaked at 101.2 % at a nominal test item concentration of 10.0 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.:

0.066 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.:

0.044 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:

EC50

Effect conc.:

0.049 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.034 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.034 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Details on results:

- Morphology evaluation: The cells were considered normal for the control and up to and including a nominal test item concentration of 0.122 mg/L. No cells were observed at nominal test item concentrations of 0.391 - 4.00 mg/L.
- Physicochemical Data: pH and temperature during the test, light intensity were within the range specified by OECD 201.
- Chemical analysis of test item concentrations: see details in the section 'Any other information on results'.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: growth rate 0.971 mg/L, yield 0.692 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 Jonckheere Terpstra test for growth and Dunnetts t-test for yield (left-sided, p<0.05)

Any other information on results incl. tables

Non-GLP range-finding test:

After 72 h, at termination of the test, a concentration response relation was observed for the inhibition of growth rate and yield from nominal test item concentrations of 0.0100 to 10.0 mg/L. The inhibition of growth rate peaked at 160.1 % and the inhibition of yield peaked at 101.2 % at a nominal test item concentration of 10.0 mg/L

Main study - Biological results:

Individual cell numbers：

Nominal concentration (mg/L)	Cell numbers (10^4/mL)
	0h	24h	48h	72h
Control	0.51	1.75	5.77	19.48
0.019	0.51	1.56	5.66	18.91
0.0381	0.51	1.44	4.79	17.61
1.222	0.51	0.85	2.14	7.00
0.391	0.51	0.35	0.67	2.35
1.25	0.51	0.17	0.29	0.4
4.00	0.51	0.07	0.08	0.06

Percentage inhibition of growth rate and yield:

Nominal concentration (mg/L)	% inhibition growth rate			% inhibition yield
	0h-24h	0h-48h	0h-72h	0h-24h	0h-48h	0h-72h
Control	0	0	0	0	0	0
0.019	9.4	0.6	0.8	15.3	2.1	3
0.0381	16	7.8	2.8	25	18.6	9.9
1.222	59.8	41.4	28.4	72.6	69	65.8
0.391	134	89.7	60.7	112.9	97	90.3
1.25	199.6	136.3	106.8	127.4	104.2	100.6
4.00	266.2	175.3	161.3	135.5	108.2	102.4

Analytical results:

Determined concentration of test item:

Nominal test concentration (mg/L)	Sampling	Measured test item concentration (mg/L)	Geometric mean concentration (mg/L)
Control	0h, fresh	< LOD	-
	24h, aged	< LOD
	48h, aged	< LOD
	72h, aged	< LOD
0.0119	0h, fresh	< LOQ	-
	24h, aged	< LOD
	48h, aged	< LOD
	72h, aged	< LOD
0.0381	0h, fresh	< LOQ	0.339
	24h, aged	< LOQ
	48h, aged	< LOD
	72h, aged	< LOD
0.122	0h, fresh	0.124	0.0555
	24h, aged	< LOQ
	48h, aged	< LOQ
	72h, aged	< LOD
0.391	0h, fresh	0.313	0.0708
	24h, aged	< LOQ
	48h, aged	< LOQ
	72h, aged	< LOD
1.25	0h, fresh	1.21	0.111
	24h, aged	< LOQ
	48h, aged	< LOQ
	72h, aged	< LOQ
4.00	0h, fresh	3.64	0.492
	24h, aged	0.255
	48h, aged	0.223
	72h, aged	0.297

Geometric means were calculated from measured concentration of samples at start, 24h, 48h and 72h.

Nominal concentration 0.0119 mg/L: since all tested concentrations were < LOQ or LOD, the concentration is not crucial for the probit analysis and geometric mean was not calculated.

For other concentrations following approach was taken: As prescribed in OECD 23 for difficult to test substances, half of the LOQ value should be employed for calculation of geometric mean (i.e. 0.05 mg/L in this study).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibitory effects were observed, with a dose response relation observed in the range 0.0119 to 4.00 mg/L. The EC10 for growth rate was set at 0.0438 mg/L (measured, geometric mean) and for yield at 0.0491 mg/L (measured, geometric mean). The EC50-value for growth rate (ErC50) was determined to be 0.0659 mg/L (measured, geometric mean). The EC50-value for yield (EyC50) was 0.0491 mg/L (measured, geometric mean).

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 10, 5, 1.00, 0.100, 0.0100 mg/L and control, a static main test with nominal test item concentrations of 4.00, 1.25, 0.391, 0.122, 0.0381, 0.0119 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 UV analysis. The measured initial concentrations of test item in the samples taken at test start were within 80 – 120 %. Afterwards, the concentrations dropped rapidly, often to below LOQ or LOD. As result, the toxicological endpoints were evaluated using the geometric mean of the measured concentrations.

Significant inhibitory effects were determined for growth rate and for yield at test item concentrations of 0.122 mg/L (nominal) and 0.0555 mg/L (measured) and above. The EC10 for growth rate was set at 0.0438 mg/L (measured, geometric mean) and for yield at 0.0491 mg/L (measured, geometric mean). The EC50-value for growth rate (ErC50) was determined to be 0.0659 mg/L (measured, geometric mean). The EC50-value for yield (EyC50) was 0.0491 mg/L (measured, geometric mean).