Thiocyanic acid, (1,3,8,10-tetrahydro-1,3,8,10-tetraoxoanthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-2,9-diyl)di-3,1-phenylene ester, reaction products with sodium sulfide (Na2(Sx))

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:

2019-12-06 to 2020-02-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23rd March, 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

01, March 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, Series On Testing And Assessment OECD No.23 (Second Edition), 08 February 2019

Version / remarks:

08, February 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 5 mg/L
- Sampling method: For determination of the test item concentration 2x six replicate samples (25 mL per replicate) from the test concentration and six replicate samples (25 mL per replicate) from the control group were taken at the start and at the end of the test in duplicates.

Vehicle:

no

Details on test solutions:

The test item is poorly soluble in the test medium; therefore preparation of test solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). In order to achieve the maximum solubility level, the test solution was prepared as follows: a supersaturated solution (5 mg/L nominal loading) was prepared by dispersing/dissolving 0.0053 g test item amount into 1060 mL of the test medium (OECD medium) and handled in ultrasonic bath for approximately 10 minutes. This solution was mixed thoroughly for a period of 24 hours to achieve an equilibrated concentration and then filtrated through a membrane filter* (0.45 µm) to separate the possible non-dissolved test material. After the formulation procedure algal cells were immediately introduced into test solution (start of the experiment).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source: Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
ACCLIMATION
- Acclimation period: no
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.5 °C - 22.7 °C in the flask
21.4 – 23.4°C within the climate chamber

pH:

7.73 – 8.29

Nominal and measured concentrations:

Nominal concentration: 5 mg/L
Measured concentartion: below LOQ and LOD

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed
- Material, size, fill volume: glass, 250 mL volume, 100 mL test medium
- Initial cells density: 104 cells/mL
- No. of organisms per vessel: 0.1 mL algal biomass (107 algal cells per mL)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium according to OECD 201

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 5046 lux, fluorescent lamps (with a spectral range of 400-700 nm)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
- Other: The cell morphology was examined in parallel.

TEST CONCENTRATIONS
- Justification for using fewer concentrations than requested by guideline: analytical method validation with 5 mg/L (showing that the solubility is below 0.05 mg/L)
- Test concentrations: 5 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.05 mg/L (LOD)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.05 mg/L (LOD)

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: > 0.05 mg/L (LOD)

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > 0.05 mg/L (LOD)

Results with reference substance (positive control):

- Results with reference substance valid? yes

The date of the latest study with the reference item Potassium dichromate was: 23 – 26 September 2019, the following results were obtained:

The 72h ErC50: 0.80 mg/L, (95 % confidence limits: 0.49 – 1.49 mg/L)
The 72h EyC50: 0.50 mg/L, (95 % confidence limits: 0.39 – 0.63 mg/L)

Reported statistics and error estimates:

Mean values and standard deviations were calculated for each concentration at each observation period using Microsoft Excel for Windows software.

For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentration were tested on significant differences to the control values using Independent Sample T-Test (α = 0.05) by statistical software program SPSS PC+.

Table 1: Algal Cell Concentrations (104 cell/ mL) during the Test Period of 72 Hours and Yield

Concentration [mg/L]		Number of Cells (× 10^4/mL)				Yield
		0 h	24 h	48 h	72 h
Control		1	2	13	36	35
		1	2	9	41	40
		1	2	13	35	34
		1	4	12	38	37
		1	3	11	42	41
		1	2	11	33	32
	Mean	1.00	2.50	11.50	37.50	36.50
	SD	0.00	0.8	1.5	3.5	3.5
WAF*		1	2	15	40	39
		1	1	14	41	40
		1	2	13	37	36
		1	2	10	32	31
		1	2	11	33	32
		1	2	16	35	34
	Mean	1.00	1.83	13.17	36.33	35.33
	SD	0.00	0.4	2.3	3.7	3.7

*WAF = water accommodated fraction (OECD No. 23.); loading rate: 5 mg/L nominal

Table 2: The Specific Growth Rates during the Test Period of 72 Hours

Concentration [mg/L]		Growth Rates (µ)
		0 – 24 h	0 – 48 h	0 – 72 h
Control		0.0289	0.0534	0.0498
		0.0289	0.0458	0.0516
		0.0289	0.0534	0.0494
		0.0578	0.0518	0.0505
		0.0458	0.0500	0.0519
		0.0289	0.0500	0.0486
	Mean	0.0365	0.0507	0.0503
	SD	0.0124	0.0029	0.0013
WAF*		0.0289	0.0564	0.0512
		0.0000	0.0550	0.0516
		0.0289	0.0534	0.0502
		0.0289	0.0480	0.0481
		0.0289	0.0500	0.0486
		0.0289	0.0578	0.0494
	Mean	0.0241	0.0534	0.0498
	SD	0.0000	0.0052	0.0006

*WAF = water accommodated fraction (OECD No. 23.); loading rate: 5 mg/L nominal

Table 3: Growth Rates (µ) and Percentage Inhibition of µ during the Test Period

Concentration [mg/L]	Growth Rates (µ) and % Inhibition of µ
	0–24 h		0–48 h		0–72 h
	µ	%	µ	%	µ	%
Control	0.0365	-	0.0507	-	0.0503	-
WAF*	0.0241	34.08	0.0534	-5.32**	0.0498	0.89

*WAF = water accommodated fraction (OECD No. 23.); loading rate: 5 mg/L nominal

**Negative value indicates increase in comparison to the control

Table 4: Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentration [mg/L]	Yield (y) and % Inhibition of y
	0–72 h
	y	%
Control	36.50	-
WAF*	35.33	3.20

Table 5: Summary of the Biological Endpoints

Endpoints (0-72 h)	Growth rate (µ) [mg/L]	Yield (y) [mg/L]
NOEC	0.05 (LOD) 5 (nominal)	0.05 (LOD) 5 (nominal)
LOEC	> 0.05 (LOD) > 5 (nominal)	> 0.05 (LOD) > 5 (nominal)

Validity criteria fulfilled:

yes

Conclusions:

In the 72-hour growth inhibition study on algae (Raphidocelis subcapitata) performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) including a loading rate of 5 mg/L, the obtained results showed that the test item had no significant toxic effects on the test system.
The 72-h NOEC was determined to be 0.05 mg/L (Limit of detection, nominal 5 mg/L).
The 72-h LOEC was determined to be > 0.05 mg/L (Limit of detection, nominal > 5 mg/L)

Executive summary:

The purpose of the study was to determine the toxicity of the test item on the growth of a unicellular green algal species in a Growth Inhibition test according to OECD 201. For this purpose, exponentially growing cultures of Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) were exposed to the saturation concentration of the test item (equivalent to 5 mg/L nominal concentration) in a limit test over a period of 72 hours, thus, over several algal generations. Because of low solubility of the test item, test solution was prepared by utilizing the water accommodated fraction (WAF) approach. The test design included six replicates at the test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10 ⁴ cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. For determination of the test item concentration, samples were taken from the test item treated group and control at the start and end of the test. The test item concentration was determined using UV/VIS spectrophotometric method. Nominal concentration of 5 mg/L was investigated in a limit test. Concurrent control ran. The measured test item concentration was below the limit of detection (LOD) over the test period of 72 hours, therefore the biological results are based on the nominal test concentration and the LOD, in line with ECHA 2017. All validity criteria were met and therefore the study can be considered as valid. All biological results are based on the nominal concentration and the analytical method’s limit of detection. In the 72-hour growth inhibition study on algae (Raphidocelis subcapitata) performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) including a loading rate of 5 mg/L, the obtained results showed that the test item had no significant toxic effects on the test system.

The 72-h NOEC was determined to be 0.05 mg/L (Limit of detection, nominal 5 mg/L).

The 72-h LOEC was determined to be > 0.05 mg/L (Limit of detection, nominal > 5 mg/L).

Description of key information

In the 72-hour growth inhibition study on algae (Raphidocelis subcapitata) performed using the OECD 201 and WAF method (according to OECD Series on Testing and Assessment No. 23) including a loading rate of 5 mg/L, the obtained results showed that the test item had no significant toxic effects on the test system.

The 72-h NOEC was determined to be 0.05 mg/L (limit of detection, nominal 5 mg/L).

The 72-h LOEC was determined to be > 0.05 mg/L (limit of detection, nominal > 5 mg/L).

 

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

5 mg/L

Additional information

The purpose of the study was to determine the toxicity of the test item on the growth of a unicellular green algal species in a Growth Inhibition test according to OECD 201. For this purpose, exponentially growing cultures of Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) were exposed to the saturation concentration of the test item (equivalent to 5 mg/L nominal concentration) in a limit test over a period of 72 hours, thus, over several algal generations. Because of low solubility of the test item, test solution was prepared by utilizing the water accommodated fraction (WAF) approach. The test design included six replicates at the test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10 ⁴ cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. For determination of the test item concentration, samples were taken from the test item treated group and control at the start and end of the test. The test item concentration was determined using UV/VIS spectrophotometric method. Nominal concentration of 5 mg/L was investigated in a limit test. Concurrent control ran. The measured test item concentration was below the limit of detection (LOD) over the test period of 72 hours, therefore the biological results are based on the nominal test concentration and the LOD, in line with ECHA 2017. All validity criteria were met and therefore the study can be considered as valid. All biological results are based on the nominal concentration and the analytical method’s limit of detection. In the 72-hour growth inhibition study on algae (Raphidocelis subcapitata) performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) including a loading rate of 5 mg/L, the obtained results showed that the test item had no significant toxic effects on the test system.

The 72-h NOEC was determined to be 0.05 mg/L (Limit of detection, nominal 5 mg/L).

The 72-h LOEC was determined to be > 0.05 mg/L (Limit of detection, nominal > 5 mg/L).