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Ecotoxicological information

Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 August 2020 - 06 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study is conducted in accordance with the relevant OECD test guideline and GLP. All validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
2019
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples for possible analysis were taken from the limit concentration and the control.
- Sampling method: Samples (volume of 1.0 mL) were taken at t=0 h and t=96 h
- 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. Lower test concentrations were prepared by subsequent dilutions of the highest test concentration in test medium. All test solutions were clear and colourless at the end of the preparation procedure.
- Eluate: N/A
- Differential loading: N/A
- Controls: Test medium without test item or other additives.
- 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: N/A, single test concentration (limit test)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported
- Other relevant information:n N/A
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Strain: ‘De Haar Vissen’, Zodiac proefacc.
- Source: Wageningen University & Research, The Netherlands
- Age at study initiation (mean and range, SD): Not reported
- Length at study initiation (length definition, mean, range and SD):
Range-finding test: 2.8 ± 0.1 cm
Limit test: 2.9 ± 0.2 cm
- Weight at study initiation (mean and range, SD):
Range-finding test: 0.26 ± 0.05 g
Final test: 0.23 ± 0.07 g

- Method of breeding: N/A
- Maintenance of the brood fish: N/A

ACCLIMATION
- Acclimation period: At least 12 days after delivery
- Acclimation conditions (same as test or not): Test medium.
During holding; conductivity, pH, nitrate, nitrite and ammonia concentration wre measured once a week. Temperature was continuously recorded. In addition, pH and temperature were measured before transferring the fish to the test system.
- Type and amount of food during acclimation: Pelleted fish food (F-0.5 GR Pro Aqua Brut, Skretting, Fontaine-lès-Vervins, France)
- Feeding frequency during acclimation: Daily until at the latest 24 hours before the start of exposure.
- Health during acclimation (any mortality observed): In the batch of fish used for the test, mortality during the 7 days prior to start of test was less than 5%.

FEEDING DURING TEST (as applicable)
- Food type: N/A
- Amount: N/A
- Frequency: No feeding from 24 hours prior to the test and during the total exposure period.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Mortality: At 2, 24, 48, 72 and 96 h
Hardness:
Expressed as CaCO3: 180 mg/L
Test temperature:
20 - 21 °C
pH:
7.1 - 7.7
Dissolved oxygen:
5.6 - 9.3 mg/L
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Limit concentration of 100 mg/L (nominal); geometric mean measured concentration of n-phenyl-diethanolamine of 70 mg/L (geometric mean concentration).

Details on test conditions:
TEST SYSTEM
- Test vessel: 10 L glass vessel
- Material, size, headspace, fill volume: All-glass, 10 L containing 8 L of test solution
- Aeration: Test solutions were not aerated during the test
- 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: 7
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: 0.20 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

- 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
- Culture medium different from test medium: No
- Intervals of water quality measurement: Dissolved oxygen content, pH and temperature: Daily in all vessels with surviving fish, beginning at the start of the test (day 0). Temperature was additionally continuously measured in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h
- Light intensity: 411 - 486 lux (The range-finding test was performed under dimmed light conditions, but the limit test was not)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Mortality and clinical effects: At 2, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 to observe for any dead or severely distressed fish. Dead fish were removed when observed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/A, limit test
- Justification for using less concentrations than requested by guideline: N/A
Range finding study:
- Test concentrations: 1.0, 10 and 100 mg/L of the parent (nominal), corresponding to 0.71, 7.2 and 71 mg/L (measured; hydrolysis product)
- Results used to determine the conditions for the definitive study - range-finding test: No mortality or clinical effects were observed in the range-finder, therefore a limit test was performed. Concentrations remained stable during the exposure period, i.e. were at 95-99% relative to the initial concentrations at the end of the test. No peaks related to the parent test item could be detected in any of the samples (samples taken from all test concentrations), indicating that the parent had been completely hydrolysed.
Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- Three fish per concentration were exposed to nominal concentrations of 1.0, 10 and 100 mg/L.
- Dissolved oxygen concentrations, pH and temperature were only measured in the lowest concentration for the final test
- Preparation of test solutions and the exposure phase was performed under dimmed light conditions due to the expected light sensitive nature of the test item.
(In the limit test, preparation of test solutions and the exposure phase was not performed under dimmed light conditions. This deviates from the handling procedures for the parent item specified in the Study Plan. However, the test item hydrolyses quickly. Consequently, the hydrolysis product was tested. This study plan deviation is considered not to have affected the outcome or integrity of the study because it did not noticeably affect the concentrations of the hydrolysis product compared to the range-finding test during which the light was dimmed.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 70 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: hydrolysis product n-phenyl-diethanolamine
Basis for effect:
mortality (fish)
Details on results:
- Other abnormalities: None observed
- Observations on body length and weight: Not reported
- Other biological observations: None observed
- Mortality of control: No mortality
- Other adverse effects control: None observed
- Hatching rate control and treatment groups (embryo): N/A
- Abnormal responses: N/A
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None observed
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
Not relevant, limit test with single concentration conducted.
Sublethal observations / clinical signs:

Validity of test

  1. No mortality was observed in the control throughout the test.
  2. The dissolved oxygen concentration was at least 60 % of the air saturation value throughout the test (> 5 mg/L at 22 °C).
  3. Test concentrations have been analytically measured.

Analysis

No peaks related to the parent test item could be detected in any of the samples, indicating that the parent had been completely hydrolysed.

The measured concentration of the hydrolysis product was 65 mg/L at the start of the test in the nominal 100 mg/L test solution of the parent. The concentration remained stable throughout the exposure period, i.e. was at 116% relative to the initially measured concentration at the end of the test.

Concentrations of the hydrolysis product were detected in the samples taken from the control but considered to derive from contamination of dimethylsulfoxide (DMSO) used for pre-treatment of the samples as similar responses were present in the blank Quality Control (QC) samples and analytical blanks. Hence, these responses were assumed not to be present in the test solutions.

Based on these results, the geometric mean concentration of the hydrolysis product measured at the limit concentration was calculated to be 70 mg/L and used to express effect parameters (see Table 2).

 

Table 1. Incidence of Mortality and Total Mortality in the Limit Test

n-phenyl-diethanolamine*

Geometric mean conc. (mg/L)

Initial Number
of fish

Cumulative mortality

Total Mortality (%)

2h

24h

48h

72h

96h

Control

7

0

0

0

0

0

0

70

7

0

0

0

0

0

0

*Hydrolysis product

Table 2. Effect Parameters

Parameter

n-phenyl-diethanolamine*

Geometric mean measured concentration (mg/L)

24h, 48h, 72h & 96h-LC50

>70

* Hydrolysis product

Table 3.pH-values and Dissolved Oxygen Concentrations (mg/L) in the Limit Test

n-phenyl-diethanolamine*

Geometric mean measured conc. (mg/L)

Day 0

Day 1

Day 2

Day 3

Day 4

pH**

O2

pH

O2

pH

O2

pH

O2

pH

O2

Control

7.6

9.3

7.6

8.0

7.7

8.2

7.4

7.4

7.5

7.8

70

7.6

9.3

7.6

8.1

7.7

8.1

7.4

7.4

7.1

5.6

*Hydrolysis product,  ** pH of culture medium was 7.9

Table 4. Temperatures (°C) Measured in the Limit Test

N-Phenyl-diethanolamine*

Geometric mean measured conc. (mg/L)

Day 0**

Day 1

Day 2

Day 3

Day 4

Control

20

21

21

21

21

70

20

21

21

21

21

* Hydrolysis product, ** Temperature of culture medium was 22 °C. Fish were gradually acclimatised to a temperature of 21 °C prior to introduction to the test solutions.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the test item did not cause any clinical effects or mortality at the limit concentration tested (100 mg/L nominal; 70 mg/L geometric mean measured). Therefore the 96 h LC50 was concluded to be > 70 mg/L (geometric mean measured).
Executive summary:

A study was performed in accordance with OECD 203 (2019), in order to determined the short-term toxicity potential of the test item to fish.

The test item hydrolyses quickly in test medium. Based on preliminary data, and in accordance with OECD GD 23, the hydrolysis product was tested instead. 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.

A limit test was performed based on the results of a range-finding study (whereby 1.0, 10 and 100 mg/L nominal concentrations were used; equating to 0.71, 7.2 and 71 mg/L measured concentrations respectively). No mortality or clinical effects were observed in the three concentrations used in the range-finder and so a limit test was performed.

For the limit test, carp (Cyprinus carpio) were exposed to a nominal concentration of 100 mg/L, for an exposure duration of 96 h (7 fish in 5 replicate vessels). The geometric mean measured concentration of n-phenyl-diethanolamine was 70 mg/L.

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.

The measured concentration of the hydrolysis product was 65 mg/L at the start of the test in the nominal 100 mg/L test solution of the parent. The concentration remained stable throughout the exposure period, i.e. was at 116% relative to the initially measured concentration at the end of the test. Based on these results, the geometric mean concentration of the hydrolysis product measured at the limit concentration was calculated to be 70 mg/L and used to express effect parameters.

A blank control was run concurrently. The validity criteria outlined in the test guideline were fulfilled. No mortality or clinical effects were observed in the control or at the limit concentration throughout the exposure period.  Therefore, 96 h LC50 was concluded to be > 70 mg/L (geometric mean measured).

Description of key information

OECD 203, Augusiak (2020), LC50: > 70 mg/L (geo. mean meas.)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
LC50
Effect concentration:
> 70 mg/L

Additional information

An study was performed in accordance with OECD 203 (2019), in order to determined the short-term toxicity potential of the test item to fish.


The test item hydrolyses quickly in test medium. Based on preliminary data, and in accordance with OECD GD 23, the hydrolysis product was tested instead. Test solutions were prepared by 3 h of magnetic stirring to allow for complete hydrolysis of the test item.


A limit test was performed based on the results of a range-finding study (whereby 1.0, 10 and 100 mg/L nominal concentrations were used; equating to 0.71, 7.2 and 71 mg/L measured concentrations respectively). No mortality or clinical effects were observed in the three concentrations used in the range-finder and so a limit test was performed.


For the limit test, carp (Cyprinus carpio) were exposed to a nominal concentration of 100 mg/L, for an exposure duration of 96 h (7 fish in 5 replicate vessels). The geometric mean measured concentration of n-phenyl-diethanolamine was 70 mg/L.


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. No peaks related to the parent test item could be detected in any of the samples, indicating that the parent had been completely hydrolysed.


The measured concentration of the hydrolysis product was 65 mg/L at the start of the test in the nominal 100 mg/L test solution of the parent. The concentration remained stable throughout the exposure period, i.e. was at 116% relative to the initially measured concentration at the end of the test. Based on these results, the geometric mean concentration of the hydrolysis product measured at the limit concentration was calculated to be 70 mg/L and used to express effect parameters.


A blank control was run concurrently. The validity criteria outlined in the test guideline were fulfilled. No mortality or clinical effects were observed in the control or at the limit concentration throughout the exposure period.  Therefore, 96 h LC50 was concluded to be > 70 mg/L (geometric mean measured).