Propanenitrile, 3-[[4-[[5,6(or 6,7)-dichloro-2-benzothiazolyl]azo]phenyl]ethylamino]-

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:

22 Nov - 26 Nov 2021

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:

adopted April 13, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control were collected at the end of the test (after 48 hours) by pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of 2 with acetonitrile.
Additional samples of the untreated controls and the sample dilution solvent were taken at each sampling test start and test end without any sample treatment.

- Sample storage conditions before analysis: deep frozen

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test item is not well soluble in test medium. To avoid physical effects of undissolved test item on the daphnids, no concentrations above the solubility limit of the test item in test water was tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 100 mg test item in 1000 mL test water. The stock suspension was stirred for 24 hours at room temperature in the dark to dissolve as much test item as possible. Then, non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter). Prior to filtration, the filter was pre-saturated with the stock solution. The solution with dissolved test item was used as the test medium of the highest test concentration and to prepare the desired 1:2, 1:4, 1:8 and 1:16 dilutions.
The test media were prepared just before introduction of the daphnids (= start of the test).
In the control, test water was used without addition of the test item.
Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:2, 1:4, 1:8 and 1:16 of this filtrate were tested, corresponding to nominal loading levels of 100, 50, 25, 12.5 and 6.3 mg test item/L. Additionally, a control was tested in parallel.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Daphnia magna (Straus), clone 5
Age at Test Start: From 2.0 to 16.5 hours old
Source: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.
The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
Feeding during the test: none
Acclimatisation: was not necessary, since the test was performed in the same medium as the culturing.

Test type:

static

Water media type:

other: Reconstituted water, Elendt "M4"

Limit test:

no

Total exposure duration:

48 h

Hardness:

Water Hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

Water Temperature:
19.1 to 19.7 °C at test start;
19.7 to 19.8 °C at test end

pH:

7.9 to 8.0 at test start;
7.8 to 7.9 at test end; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

7.3 to 9.1 mg/L at test start;
7.6 at test end

Nominal and measured concentrations:

nominal: Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:2, 1:4, 1:8 and 1:16 of this filtrate were tested, corresponding to nominal loading levels of 100, 50, 25, 12.5 and 6.3 mg test item/L. Additionally, a control was tested in parallel.

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal values since the concentrations of the test item were within ± 20% of the nominal concentrations during the test.

Details on test conditions:

Test Vessel: Glass beakers of 100 mL volume containing approximately 60 mL of test medium. The test vessels were covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 580 to 630 lux (measured once during the test).
Introduction of Daphnids: 20 daphnids per control and test concentration, divided into 4 groups of 5 animals, each group in one test vessel.
Replicates: The test was performed with four replicates per treatment group.
Exposure Time: 48 hours
Test Procedure: A static test was performed.
Feeding: None
Sampling: Samples were taken at test start (0 hours) and test end (48 hours)

Test Parameter:
Immobility: The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Those animals that are not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile (even if they could still move their antennae).

Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

42.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

26.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Signs of Intoxication after 48 Hours:
After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the nominal test item level of 25 mg test item/L. At the nominal loading level of 50 mg test item/L, 16 daphnids were immobile and all daphnids were immobile at the nominal loading level of 100 mg test item/L.
At the nominal loading level 50 mg/L 4 Daphnia had agglutinated antennae.

Results with reference substance (positive control):

Results of the most recent Test with the Reference Item Potassium Dichromate
Dose response test: 0.40, 0.60, 0.89, 1.33 and 2.0 mg/L
EC50 24h: 1.21 mg/L
NOEC 24 h: 0.60 mg/L
LOEC 24 h: 0.89 mg/L
EC50 48 h: 0.844 mg/L
NOEC 48 h: 0.40 mg/L
LOEC 48 h: 0.60 mg/L

The reference test was valid; The results indicate that the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).

Reported statistics and error estimates:

The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by weibull analysis.
The NOEC and LOEC after 24 hours were calculated by Fisher´s exact binominal test with Bonerroni corrections and the NOEC and LOEC after 48 hours were calculated by Step-down Cochran Armitage test procedure.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Validity criteria

Criterion	value required	value obtained	fullfilled
Control immobilisation rate	< 10%	0%	yes
Dissolved oxygen concentration	at the end of the test >= 3 mg O2/L	≥ 7.6 mg O2/L in in all treatment groups at the end of the test	yes

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of the test item Disperse Red 153 to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEL was determined to be 25.0 mg test item/L. The 48-hour LOEL was determined to be 50.0 mg test item/L and the 48-hour EL50 value was calculated to be 42.2 mg test item/L.
The initial concentrations and the exposure concentrations during the test were determined in the analytical part.
Since no quantifiable amount of the test item was observed in any of the test samples, the results are reported based on the nominal loading levels.

Executive summary:

Test Species: Daphnia magna, clone 5; 2.0 to 16.5 hours old

Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) each containing 20 individuals. The mobility of the daphnids was determined in a static 48-hour test by visual observation after 24 and 48 hours. 

Endpoints: Number of immobile organisms after 24 and 48 hours

Test Concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16) and a control.

Test Conditions: Water temperature: 19.1 to 19.8 °C; pH value: 7.8 to 8.0; dissolved oxygen concentration: 7.3 to 9.1 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 580 to 630 lux; and thus were within the ranges requested by guideline OECD 202

Results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the nominal test item level of 25 mg test item/L. At the nominal loading level of 50 mg test item/L, 16 daphnids were immobile and all daphnids were immobile at the nominal loading level of 100 mg test item/L.

At the nominal loading level 50 mg/L 4 Daphnia had agglutinated antennae.

Parameter
	after 24 h	after 48 h
EC50 mg test item/L	89.6	42.2
EC20 mg test item/L	86.4	32.1
EC10 mg test item/L	84.3	26.8
NOEL mg test item/L	50.0	25.0
LOEL mg test item/L	100.0	50.0

Analytical Results:

The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. No signal above the Limit of Quantification (LOQ) of 0.075 mg test item/L was observed in any of the test samples.

Conclusion:

The toxic effect of the test item to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEL was determined to be 25.0 mg test item/L. The 48-hour LOEL was determined to be 50.0 mg test item/L and the 48-hour EL₅₀ value was calculated to be 42.2 mg test item/L.

The initial concentrations and the exposure concentrations during the test were determined in the analytical part.

Since no quantifiable amount of the test item was observed in any of the test samples, the results are reported based on the nominal loading levels.

Description of key information

The toxic effect of the test item Disperse Red 153 to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEL was determined to be 25.0 mg test item/L. The 48-hour LOEL was determined to be 50.0 mg test item/L and the 48-hour EL50 value was calculated to be 42.2 mg test item/L.
The initial concentrations and the exposure concentrations during the test were determined in the analytical part.
Since no quantifiable amount of the test item was observed in any of the test samples, the results are reported based on the nominal loading levels.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

expressed as loading level

Effect concentration:

42.2 mg/L

Additional information

The test substance was assessed for toxicity to Daphnia magna. The study encompassed 6 treatment groups (5 dose rates of the test item and a control) each containing 20 individuals. The mobility of the daphnids was determined in a static 48-hour test by visual observation after 24 and 48 hours. The number of immobile organisms was evaluated after 24 and 48 hours.

Tested concentrations were 100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16) and a control. The water temperature was 19.1 to 19.8 °C; pH value: 7.8 to 8.0; dissolved oxygen concentration: 7.3 to 9.1 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 580 to 630 lux; and thus were within the ranges requested by guideline OECD 202

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the nominal test item level of 25 mg test item/L. At the nominal loading level of 50 mg test item/L, 16 daphnids were immobile and all daphnids were immobile at the nominal loading level of 100 mg test item/L.

At the nominal loading level 50 mg/L 4 Daphnia had agglutinated antennae.

Parameter
	after 24 h	after 48 h
EC50 mg test item/L	89.6	42.2
EC20 mg test item/L	86.4	32.1
EC10 mg test item/L	84.3	26.8
NOEL mg test item/L	50.0	25.0
LOEL mg test item/L	100.0	50.0

The quantification of the test item in the test samples was performed using liquid chromatography with UV detection. No signal above the Limit of Quantification (LOQ) of 0.075 mg test item/L was observed in any of the test samples. The initial concentrations and the exposure concentrations during the test were determined in the analytical part. Since no quantifiable amount of the test item was observed in any of the test samples, the results are reported based on the nominal loading levels.

The toxic effect of the test item to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEL was determined to be 25.0 mg test item/L. The 48-hour LOEL was determined to be 50.0 mg test item/L and the 48-hour EL₅₀ value was calculated to be 42.2 mg test item/L.