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))

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-30 to 2017-11-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13th April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

April 1996

Deviations:

no

Guideline:

other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000

Version / remarks:

September 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Five replicate samples were analysed from the test (saturated) solution at the start and four samples at the end of the renewal periods. Four replicate samples were analysed from the control at the start and at the end of the experiment.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For preparation of test solution, the water-accommodated fraction (WAF) approach was taken, as described in OECD Guidance Document 23. A supersaturated solution (100 mg/L nominal loading) was prepared by adding an amount of 0.03 g test item in 300 mL test medium (ISO medium). The test solution was handled by ultrasonic bath for 10 minutes, thereafter stirred for a period of 24 hours to achieve an equilibrated concentration. The test solution was then filtrated through a membrane filter (0.45 µm; Nalgene® membrane) to separate the possible non-dissolved test material.
- Controls: positive control, negative control
- Test Water (Dilution Water): Reconstituted water (ISO medium, according to OECD 202) was used as dilution water in the experiment. Separate stock solutions of individual substances were first prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE ELIX 3 water purification system). The ISO medium was prepared by adding 25 mL from each of four stock solutions to one litre deionised water.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Origin: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Breeding: The Daphnia are bred in the Laboratory of TOXI-COOP ZRT.
- Number of animals: Twenty animals in both groups (concentration and untreated control), divided into 4 concurrent batches (5 animals per batch).
- Age of animals: Less than 24 h old at the beginning of the test.
- Sex: Female
- Animal health: Apparently healthy animals were used in this test with a known history
- Acclimatization: Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test. 
- Food and Feeding: The daphnids were fed with concentrated algal suspension of Raphidocelis subcapitata during the holding. Test animals were not fed during the exposure.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Post exposure observation not performed.

Hardness:

213.6 mg/L (as CaCO3) for ISO Medium used in the study

Test temperature:

20.6 – 21.4°C

pH:

7.39 – 8.35

Dissolved oxygen:

6.87 – 8.04 mg/L

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured: 0.297 mg/L (geometric mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers
- Type: Open
- Material, size, headspace, fill volume: Glass, 50 mL, at least 4 mL test solution/animal
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Medium

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: The light-dark cycle during the test was 16 hours light (artificial illumination) and 8 hours darkness

EFFECT PARAMETERS MEASURED
Daphnids were observed for immobility or mortality by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.

RANGE-FINDING STUDY
In order to select appropriate test concentrations for use in the definitive test, non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item. Due to the test item’s low solubility, preparation of test solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) as follows: a test item stock solution was prepared by adding an amount of test item into the dilution water (ISO medium) to get the nominal concentration of 100 mg/L and was handled in ultrasonic bath for 10 minutes. The stock solution was mixed for a period of 48 hours to achieve an equilibrated concentration and then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. Untreated control ran parallel in the test. In the preliminary range-finding tests ten (2 x 5) daphnids in the test concentration and control were exposed for 48 hours. The tests were carried out under static conditions.

- Test concentrations: 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 0.297 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Concentration based on water-accommodated fraction of test item

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

> 0.297 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Concentration based on water-accommodated fraction of test item

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.297 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Concentration based on water-accommodated fraction of test item

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.297 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Concentration based on water-accommodated fraction of test item

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.297 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Concentration based on water-accommodated fraction of test item.

Basis for effect:

mobility

Details on results:

VALIDITY OF THE STUDY
Immobilisation was not observed in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was in the range of 7.27 – 7.70 mg/L (more than 3 mg/L in all cases). All validity criteria were within acceptable limits and therefore the study can be considered as valid.

ANALYTICAL RESULTS
A saturated test item solution (limit concentration) and a concurrent control were included in the main test. The concentration of the test item was analytically determined at the start and at the end of the experiment. In the treated group mean of the initial measured concentration was 0.312 mg/L. Mean measured concentration at the end of test was 0.282 mg/L, corresponding to 90 % of the initial measured test concentration. The test item was not detected in the untreated control group (i.e. signal intensities measured for the control samples were less than 20 % of the analytical quantification limit (LOQ)). The exposure concentration was calculated as the geometric mean of the start and end values and determined to be 0.297 mg/L. This concentration was considered as the saturation concentration in the test medium, based on water-accommodated fractions of the test item (equivalent to 100 mg/L nominal concentration).

BIOLOGICAL RESULTS AND PERFORMANCE OF THE TEST
Testing units were kept in a climate chamber at a temperature of 20.5 – 21.5 °C, while temperature in the test glasses ranged between 20.6 and 21.4 °C. The pH was measured as 7.39 – 8.35 and the dissolved oxygen concentration between 6.87 – 8.04 mg/L during the study. The light-dark cycle during the test was 16 hours light (artificial illumination) and 8 hours darkness. There was no immobilisation observed in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Results with reference substance (positive control):

The 24h EC50 was determined to be 1.41 mg/L (95% conf. limits: 1.19 – 1.68 mg/L), which falls within the range of 0.6 mg/L and 2.1 mg/L as given in the guideline.

Reported statistics and error estimates:

A limit test was performed and toxic effects were not observed, therefore statistical analysis was not necessary. The NOEC, LOEC and ECx values of the test item were determined directly from the raw data.

Table 1: pH-values Measured during the Experiment

Test Group	Replicate	pH
		0h	48h
Control	1	7.39	7.75
	2		8.07
	3		8.25
	4		8.22
Saturated test concentration (0.297 mg/L mean measured)	1	7.93	8.23
	2		8.23
	3		8.35
	4		8.34

 

Table 2: Dissolved Oxygen Concentrations Measured during the Experiment

Test Group	Replicate	Dissolved Oxygen Concentration[mg/L]
		0h	48h
Control	1	8.04	7.70
	2		7.27
	3		7.51
	4		7.59
Saturated test concentration (0.297 mg/L mean measured)	1	6.87	7.63
	2		7.58
	3		7.57
	4		7.53

 

Table 3: Temperatures Measured during the Experiment

Test Group	Replicate	Temperature[°C]
		0h	48h
Control	1	21.1	20.7
	2		20.6
	3		20.7
	4		20.7
Saturated test concentration (0.297 mg/L mean measured)	1	21.4	20.8
	2		20.6
	3		20.6
	4		20.7

       Table 4: Immobilization of the Test Animals

Test Group	Replicate	Number of treated animals	Number of immobilised animals
			24 h	48 h
Control	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
Saturated test concentration (0.297 mg/L mean measured)	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0

Results of the analysis

Measured concentrations ofLeuco Sulfur Red 14 in ISO medium(according to OECD 202) are summarised in Table 4.

 

Table 5. Leuco Sulfur Red 14 Concentrations Measured during the Study

Saturated solution	Analytical occasion	Mean of the measured concentrations (mg/L) with the 95% confidence intervals	Measured concentration in % of the start
	Start	0.312±0.034	-
	End	0.282±0.025	90

 The ISO control samples show a small absorbance. The amount of any interfering reagent component was less than 20 % of the lowest concentration of the calibration curve.

Validity criteria fulfilled:

yes

Conclusions:

The 48-h EC10, EC20, EC50 and LOEC of the test item are higher than the solubility level of the test item in the test medium, which corresponds to a mean measured concentration of 0.297 mg/L (100 mg/L in nominal).

Executive summary:

Acute toxicity of the test item was assessed on Daphnia magna in a GLP-compliant 48-hour Acute Immobilisation Test according to OECD Guideline 202, EU Method C.2 and US EPA OPPTS 850.1010. Daphnids were exposed to aqueous test media (ISO Medium) containing the test item at the limit concentration of 100 mg/L (nominal, test solutions prepared as water-accommodated fraction) for 48 hours in a static test. The quantification of the test item was performed by UV/VIS spectrophotometry. Samples were taken from the test concentration and the control at the start and at the end of the experiment and analysed by UV/VIS spectrophotometric method. The mean determined exposure concentration of the test item remained between 80 and 120% throughout the test in comparison to that of the measured test item concentration at the start of the test, and was 0.297 mg/L which was calculated as the geometric mean of the measured start and end concentrations. Twenty healthy, young female daphnids < 24 hours of age and with known origin (divided into 4 concurrent batches) were tested each, exposed to the saturated test concentration and in the control. The immobilisation of the test animals was recorded 24 and 48 hours after test start. In result, there was no immobilisation in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. All validity criteria were met and therefore the study can be considered as valid. In conclusion: the test item had no toxic effect at aquatic saturation (i.e.limit test concentration) on Daphnia magna; the EC10, EC20, EC50 results and the LOEC are higher than the solubility level of the test item in the test medium, which corresponds to 0.297 mg/L.

Description of key information

The 48-h EC10 and EC50 of the test item are higher than the solubility level of the test item in the test medium, which corresponds to a mean measured concentration of 0.297 mg/L (100 mg/L in nominal).

Additional information

Acute toxicity of the test item was assessed on Daphnia magna in a GLP-compliant 48-hour Acute Immobilisation Test according to OECD Guideline 202. Daphnids were exposed to aqueous test media (ISO Medium) containing the test item at the limit concentration of 100 mg/L (nominal; test solutions prepared as water-accommodated fraction) for 48 hours in a static test. The quantification of the test item was performed by UV/VIS spectrophotometry. Samples were taken from the test concentration and the control at the start and at the end of the experiment and analysed by UV/VIS spectrophotometric method. The mean determined exposure concentration of the test item remained between 80 and 120% throughout the test in comparison to that of the measured test item concentration at the start of the test, and was 0.297 mg/L which was calculated as the geometric mean of the measured start and end concentrations. Twenty healthy, young female daphnids < 24 hours of age and with known origin (divided into 4 concurrent batches) were tested each, exposed to the saturated test concentration and in the control. The immobilisation of the test animals was recorded 24 and 48 hours after test start. In result, there was no immobilisation in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. All validity criteria were met and therefore the study can be considered as valid. In conclusion: the test item had no toxic effect at aquatic saturation (i.e.limit test concentration) on Daphnia magna; the EC10,EC20,EC50 results and the LOEC are higher than the solubility level of the test item in the test medium, which corresponds to 0.297 mg/L.