Santicizer P1400

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:

2016-01-04 to 2016-02-17

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polyadd Limited; Batch no. 0900
- Expiration date of the lot/batch: 2016-10-22
- Purity test date: 2015-10-22
- Purity: 98.8

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark
- Stability under test conditions: Not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material): Clear colorless liquid

Analytical monitoring:

yes

Details on sampling:

- Concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution. Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100% v/v saturated solution
- Sampling method:

Range-finding Test:
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test:
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10, 18, 32 and 56% v/v saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

- Sample storage conditions before analysis: A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis

Vehicle:

yes

Remarks:

Reconstituted water (ISO medium)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 5.2 mg/L could be obtained using a saturated solution method of preparation.

A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 0.10, 1.0 and 10% v/v saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10, 18, 32 and 56% v/v saturated solution. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Not specified
- Justification for species other than prescribed by test guideline: Daphnia magna is a freshwater invertebrate representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems.
- Source: in house culture
- Age of parental stock (mean and range, SD): less than 24 hours old
- Feeding during test: not fed

ACCLIMATION
- Acclimation period: not specified
- Acclimation conditions (same as test or not): Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at 19 to 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Type and amount of food: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin®flake food suspension
- Feeding frequency: Daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing

Test type:

static

Water media type:

other: Reconstituted water (ISO medium)

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

48 hours

Hardness:

250 mg/L as CaCO3

Test temperature:

21 to 22 °C

pH:

7.8 ± 0.2

Dissolved oxygen:

8.6 to 8.7 mg O2/L

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56, 100 (% v/v)
Measured concentrations: 0.41, 0.76, 1.4, 2.3, 4.4 (mg/L) at 0 hours

Details on test conditions:

TEST SYSTEM
- Test vessel: Beaker
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 150 mL glass beakers
- Aeration: Not aerated
- Type of flow-through (e.g. peristaltic or proportional diluter): static
- Renewal rate of test solution (frequency/flow rate): not specified
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: Not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium)
CaCl2.2H2O 294 mg/L
MgSO4.7H2O 123 mg/L
NaHCO3 64.75 mg/L
KCl 5.75 mg/L

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value. The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

OTHER TEST CONDITIONS
- Photoperiod: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Light intensity: 586 to 634 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks:

effects on mobility observed at the 1.4 mg/L concentration

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.73 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: No effects observed

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Sub-lethal effects of exposure were observed in the 32 and 56% v/v saturated solution test concentrations. This response was reduced mobility
- Observations on body length and weight: Not specified
- Other biological observations: No effects
- Mortality of control: No
- Other adverse effects control: No
- Abnormal responses: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
No sub-lethal effects observed

Reported statistics and error estimates:

The EC50 value at 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Concentration and the No Observed Effect Concentration at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Geometric Mean Measured Test Concentrations
The geometric mean measured test concentrations of the samples were calculated as follows using the measured test concentrations of replicates R1 – R4 pooled:

GM = √C0 C1
GM = geometric mean measured test concentration (mg/L)
C0 = measured concentration at the start of the test (mg/L)
C1 = measured concentration at the end of the test (mg/L)

Table 1. Test Concentrations
Nominal Test Concentration (% v/v Saturated Solution)	0 Hour Measured Concentration (mg/L)	48 Hour Measured Concentration (mg/L)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a Percentage of the 0 Hour Measured Concentration (%)
10	0.41	0.38	0.39	95
18	0.76	0.70	0.73	96
32	1.4	1.4	1.4	100
56	2.3	1.8	2.0	87
100	4.4	3.8	4.1	93

Table 2. Cumulative Immobilization Data and Observations in the Definitive Test
Nominal Concentration (% v/v Saturated Solution)	Geometric Mean Measured Concentration (mg/L)	24 hours
		Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
		R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control		0	0	0	0	0	0	5N	5N	5N	5N
10	0.39	0	0	0	0	0	0	5N	5N	5N	5N
18	0.73	0	0	0	0	0	0	5N	5N	5N	5N
32	1.4	2	3	1	1	7	35	3R	2R	4R	4R
56	2.0	4	3	5	5	17	85	1R	2R	A.I	A.I
100	4.1	5	5	5	5	20	100	A.I	A.I	A.I	A.I

R1 – R4 = Replicates 1 to 4

N = No sub-lethal effects observed

R= Reduced mobility

A/I = All daphnia immobilized

 

Table 3. Cumulative Immobilization Data and Observations in the Definitive Test
Nominal Concentration (% v/v Saturated Solution)	Geometric Mean Measured Concentration (mg/L)	48 hours
		Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
		R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control		0	0	0	0	0	0	5N	5N	5N	5N
10	0.39	0	0	0	0	0	0	5N	5N	5N	5N
18	0.73	0	0	0	0	0	0	5N	5N	5N	5N
32	1.4	3	4	5	4	16	80	2R	1R	A.I	1R
56	2.0	5	5	5	5	20	100	A.I	A.I	A.I	A.I
100	4.1	5	5	5	5	20	100	A.I	A.I	A.I	A.I

R1 – R4 = Replicates 1 to 4

N = No sub-lethal effects observed

R= Reduced mobility

A/I = All daphnia immobilized

Validity criteria fulfilled:

yes

Remarks:

The test was considered to be valid since none of the control daphnids showed immobilization or other signs of disease/stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Conclusions:

Sub-lethal effects of exposure were observed in the 32 and 56% v/v saturated solution test concentrations. This response was reduced mobility. Based on the effects observed the 48-hour EC50 was determined to be 1.1 mg/L, while the NOEC and LOEC were determined to be 0.73 and 1.4 mg/L, respectively.

Executive summary:

A key 48-hour acute immobilization test was carried out to assess the acute toxicity of the test material (1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester; CAS# 1200806-67-2) to Daphnia magna.

 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals each) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours at a temperature of approximately 21 to 22°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.41 to 4.4 mg/L. Analysis of the test preparations at 48 hours showed measured test concentrations had declined, to between 0.38 and 3.8 mg/L (76% to 102% of the 0-Hour measured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case”

analysis of the data.

 

Sub-lethal effects of exposure were observed in the 32 and 56% v/v saturated solution test concentrations. This response was reduced mobility. Based on the effects observed the 48-hour EC₅₀ was determined to be 1.1 mg/L, while the NOEC and LOEC were determined to be 0.73 and 1.4 mg/L, respectively.

Description of key information

A key 48-hour acute immobilization test was carried out to assess the acute toxicity of the test material (1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester; CAS# 1200806-67-2) to Daphnia magna.

 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals each) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution for 48 hours at a temperature of approximately 21 to 22°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.41 to 4.4 mg/L. Analysis of the test preparations at 48 hours showed measured test concentrations had declined, to between 0.38 and 3.8 mg/L (76% to 102% of the 0-Hourmeasured test concentrations) and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

 

Sub-lethal effects of exposure were observed in the 32 and 56% v/v saturated solution testconcentrations. This response was reduced mobility. Based on the effects observed the 48-hour EC₅₀ for P1400 was determined to be 1.1 mg/L, while the NOEC and LOEC were determined to be 0.73 and 1.4 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

immobilisation

Effect concentration:

1.1 mg/L