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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 August 2020 - 01 October 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted in accordance with the relevant OECD test guideline and according to GLP. All validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 April 2004
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples for possible analysis were taken from all test concentrations and the control.
- Sampling method: Samples (at a volume of 1.0 mL from the approximate centre of the test vessels) were taken at t=0 h and t=48 h. At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Since the test item was shown to be hydrolytically unstable (see Sec 5.1.2. Hydrolysis record in this data set, Test Facility Reference No. 20194879), the method of preparation was based on the principles laid down in the OECD Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals.
In Test Facility Reference No. 20194879, it was confirmed that the half-life time of the test item was below 1 hour in aqueous solutions at pH values relevant for the present study. It was therefore decided, in agreement with OECD GD 23, to test the resulting hydrolysis product. In Test Facility Reference No. 20194899 (see Sec 8 Analytical methods record in this data set), it was additionally confirmed that in aqueous solutions the reaction product of n-phenyl-diethanolamine with a single molecule of formaldehyde is converted back to n-phenyl-diethanolamine.
Based on these results, it was concluded to prepare nominal concentrations of the parent test item in test medium, mix the solution sufficiently long for complete hydrolysis, and to use the resulting solutions in the further tests. Analytical measurements were performed for both components, i.e. the parent test item n-phenyl-diethanolamine, reaction products with formaldehyde and the hydrolysis product n-phenyl-diethanolamine.

The preparation of test solutions started with the highest concentration of 100 mg/L applying a three-hour period of magnetic stirring to ensure maximum hydrolytic conversion of the test item. After the three-hour period of magnetic stirring the lower test concentrations were prepared by subsequent dilutions of the highest test concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
Due to indicated light sensitivity, glassware was wrapped in aluminum foil and preparation of test solutions was performed under dimmed light conditions to minimise exposure to light.
- Eluate: N/A
- Differential loading: N/A
- Controls: Blank control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Test concentration separation factor: 1.8
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported
- Other relevant information: N/A
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: Straus, 1820; at least 3rd generation, obtained by a cyclical parthenogenesis under specified breeding conditions
- Justification for species other than prescribed by test guideline: N/A
- Age at study initiation (mean and range, SD): Newborn daphnids, < 3 d old
- Weight at study initiation (mean and range, SD): Not reported
- Length at study initiation (length definition, mean, range and SD): Not reported
- Stage and instar at study initiation: Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old
- Method of breeding: Approx 250 newborn daphnids, < 3 d old were placed in 5 L of medium in an all-glass culture vessel at the start of each batch. Max age of the cultures was 4 weeks. After 7 d of cultivation, half of the medium was renewed twice a week. Culture medium: M7.
- Source: In-house laboratory culture
- Age of parental stock (mean and range, SD): < 3 d old
- Feeding during test
- Food type: Freshwater algae suspension
- Amount: Not reported
- Frequency: Daily

ACCLIMATION
N/A

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Daphnids used in the test system originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20% , presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Expressed as CaCO3: 180 mg/L
Test temperature:
20 - 21°C
pH:
7.8 - 8.0
Dissolved oxygen:
8.3 - 8.9 mg/L
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 10, 18, 32, 56 and 100 mg/L (parent test item)
Gemoetric mean measured: 6.1, 11, 18, 34, 63 mg/L (n-phenyl-diethanolamine, hydrolysis product)
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL glass vessel
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All-glass
- Volume of solution: 50 mL
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- 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): N/A
- Loading: 5 per vessel containing 50 mL of test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The preparation of test solutions started with the highest concentration of 100 mg/L applying a three-hour period of magnetic stirring to ensure maximum time for hydrolytic conversion of the test item. After the three-hour period of magnetic stirring the lower test concentrations were prepared by subsequent dilutions of the highest test concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure. Due to indicated light sensitivity, glassware was wrapped in aluminum foil and preparation of test solutions was performed under dimmed light conditions to minimise exposure to light.
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Salinity: Not reported
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control. Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Test performed in the dark
- Light intensity: N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility (including mortality): 24 and 48 h. 'Immobility' was judged as those animals not able to swim within 15 seconds after gentle agitation of the test vessel, even if they can still move their antennae.

VEHICLE CONTROL PERFORMED: N/A

COMBINED LIMIT/RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 mg/L and 100 mg/L nominal (parent test item); measured concentrations of hydrolysis product were 0.081, 0.79, 7.8 and 73 mg/L.
- Results used to determine the conditions for the definitive study: No biologically relevant immobility was observed in the three lowest test concentrations throughout the test. In the 100 mg/L test group, 70 and 100 % immobility was observed at 24 and 48 hours of exposure, respectively.
Samples taken from all test concentrations at the start and end of the test were analysed. No peaks related to the parent test item could be detected in any of the samples, indicating that the parent had been completely hydrolysed. During the exposure period, the concentrations generally remained stable, i.e. were at 80 - 98 % relative to the initial concentrations at the end of the test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: hydrolysis product, i.e. n-phenyl-diethanolamine
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
65 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: hydrolysis product, i.e. n-phenyl-diethanolamine
Basis for effect:
mobility
Remarks on result:
other: Estimated by extrapolation
Details on results:
- Behavioural abnormalities: None
- Observations on body length and weight: Not reported
- Other biological observations: N/A
- Mortality of control: None
- Other adverse effects control: None
- Immobilisation of control: No biologically relevant immobility was observed in the control
- Abnormal responses: At 24 h of exposure, three daphnids, i.e. 15 %, were observed floating at the surface of the test solution. Since at the end of the test, no daphnids were immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium, the observation made at 24 h of exposure was accepted and the test considered to be valid.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):
- Results with reference substance valid? Yes. The batch of Daphnia magna tested showed expected sensitivity to potassium dichromate based on the range specified in ISO International Standard 6341, October 2012. The raw data from this study are kept in the Charles River Den Bosch archives. The test was performed non-GLP. Study information:
Test Facility Study No. 20270039
Experimental Start Date: 21 September 2020
Experimental Completion Date: 25 September 2020
Reported statistics and error estimates:
Determination of the average exposure concentrations:
The average exposure concentrations were calculated as:
√(Ct=0 * Ct=48), being the geometric means of the concentrations of n-phenyl-diethanolamine in the samples taken at the start (Ct=0) and the end of the test (Ct=48).
The EC50 could not be determined using a regression method. Instead, the 24 and 48 h EC50 values were calculated by applying the Spearman-Karber procedure (non-linear; without trimming) on the percentages of affected daphnids and the logarithms of the corresponding geometric mean concentrations of the hydrolysed test item.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Validity criteria

  1. At 24 hours of exposure, three daphnids, i.e. 15 %, were observed floating at the surface of the test solution. Since at the end of the test, no daphnids were immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium, the observation made at 24 hours of exposure was accepted and the test considered to be valid.
  2. The dissolved oxygen concentration at the end of the test was ≥ 3 mg/L in control and test concentration vessels.

Analysis

Samples taken from all test concentrations were analysed. No peaks related to the parent test item could be detected in any of the samples throughout the study, indicating that the parent had been completely hydrolysed.

The measured concentrations for the hydrolysis product were 5.9, 11, 18, 34 and 61 mg/L in solutions prepared at nominally 10, 18, 32, 56 and 100 mgparent/L, respectively, at the start of the test. During the exposure period, the concentrations remained stable, i.e. were at 100 - 110 % relative to the initial concentrations at the end of the test. Based on these results, the geometric mean measured concentrations of n-phenyl-diethanolamine were calculated and used to express effect parameters (see Table 3).

Table 1. Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)

Replicate

n-phenyl-diethanolamine

Geometric mean measured conc. (mg/L)

Control

6.1

11

18

34

64

0

A

5

5

5

5

5

5

 

B

5

5

5

5

5

5

 

C

5

5

5

5

5

5

 

D

5

5

5

5

5

5

 

Total introduced

20

20

20

20

20

20

               

24

A

0 (2)

0

0

0 (1)

0

3

 

B

0 (1)

0 (1)

0

0

0 (1)

1

 

C

0

0 (1)

0

0

0

4

 

D

0

0

0 (1)

0

0

1

 

Total immobilised

0

0

0

0

0

9

 

Effect %

0

0

0

0

0

45

 

 

 

 

 

 

 

 

48

A

0

0

0

0

0

5

 

B

0

0

0

0

0

5

 

C

0

0

0

0

1

5

 

D

0

0

0

0

0

5

 

Total immobilised

0

0

0

0

1

20

 

Effect %

0

0

0

0

5

100

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were re-immersed into the respective solutions before recording of mobility.

Table 2. Geometric Mean Versus Nominal Concentrations

n-phenyl-diethanolamine, reaction products with formaldehyde

Nominal conc. (mg/L)

n-phenyl-diethanolamine

Measured conc. (mg/L)

n-phenyl-diethanolamine

Geometric mean measured conc. (mg/L)

t=0h

t=48h

10

5.85

6.44

6.1

18

11*

11.7

11

32

17.7

19.2

18

56

34.1

34.1

34

100

61.3

67.1

64

*estimated by extrapolation of the calibration curve

Table 3. Effect Parameters

Parameter

n-phenyl-diethanolamine

Geometric mean measured conc. (mg/L)

95%-confidence interval (mg/L)

24h-EC50

65*

57-75

48h-EC50

45

42-48

*estimated by extrapolation

Table 4. pH and Oxygen Concentrations (mg/L) During the Final Test

n-phenyl-diethanolamine

Geometric mean measured conc. (mg/L)

Start (t=0 h)

End (t=48 h)

O2

pH

O2

pH

Control

8.7

7.8

8.8

8.0

6.1

8.7

7.8

8.5

8.0

11

8.6

7.8

8.5

8.0

18

8.7

7.8

8.4

8.0

34

8.7

7.8

8.4

8.0

64

8.8

7.8

8.3

8.0

Validity criteria fulfilled:
yes
Conclusions:
In the highest test group, 45 and 100 % immobility were observed at 24 and 48 hours of exposure, respectively. The 48 h immobility EC50 based on geometric mean concentrations of the hydrolysis product (i.e. n-phenyl-diethanolamine) was determined to be 45 mg/L (95 % confidence interval: 42 - 49 mg/L).
Executive summary:

A test was conducted in accordance with OECD 202 (2004), to determine the acute toxicity potential of the test item to Daphnia magna, by examining effects on mobility.

A full test was performed based on the results of a preceding combined limit/range-finding test. Since the test item was shown to be hydrolytically unstable, the method of preparation was based on the principles laid down in the OECD Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals.

The preparation of test solutions started with the highest concentration of 100 mg/L applying a three-hour period of magnetic stirring to ensure maximum hydrolytic conversion of the test item. After the three-hour period of magnetic stirring the lower test concentrations were prepared by subsequent dilutions of the highest test concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure. Analytical measurements were performed for both components, i.e. the parent test item n-phenyl-diethanolamine, reaction products with formaldehyde and the hydrolysis product n-phenyl-diethanolamine.

In the combined limit/range-finder, 10 daphnids per concentration (in duplicate, 5 daphnids per vessel) were exposed to nominally 0.10, 1.0, 10 mg/L and 100 mg/L (parent test item; corresponding to 0.081, 0.79, 7.8 and 73 mg/L measured concentrations of the hydrolysis product).

In the final test, 20 daphnids per concentration (4 replicates, 5 daphnids per vessel) were exposed to nominal concentrations of 10, 18, 32, 56 and 100 mg/L (parent test item; corresponding to 5.9, 11, 18, 34 and 61 mg/L measured concentrations of the hydrolysis product). The test was static, conducted for an exposure duration of 48 h, at 20-21 ºC. A blank control was run concurrently in the test.

Following analytical measurements, no peaks related to the parent test item could be detected in any of the samples, indicating that the parent had been completely hydrolysed. During the exposure period, the concentrations remained stable, i.e. were at 100 - 110 % relative to the initial concentrations at the end of the test. 

At 24 h of exposure, three daphnids, i.e. 15%, were observed floating at the surface of the test solution. Since at the end of the test, no daphnids were immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium, the observation made at 24 h of exposure was accepted and the test considered to be valid. The other validity criterion (dissolved oxygen was => 3 mg/L at the end of the test, in control and test concentration vessels) was also met.

No biologically relevant immobility was observed in the control and the four lowest test concentrations throughout the test. In the highest test group, 45 and 100 % immobility were observed at 24 and 48 hours of exposure, respectively. The 24 h EC50, estimated by extrapolation, was found to be 65 mg/L (geo. mean measured; 95 % confidence interval 57-75 mg/L). The 48 h EC50 was determined to be 45 mg/L (geo. mean measured; 95 % confidence interval 42-48 mg/L).

Description of key information

OECD 202, Augusiak (2020), 48 EC50: 45 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
45 mg/L

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