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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
26 March 1996 - 28 March 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: the EU Directive 79/831/EEC, Annex V, part C, which is comparable to the OECD guideline 202.
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-ethoxyethylamin
- Physical state: fluid
- Analytical purity: 99.6 %
- Impurities (identity and concentrations): 0.11 % water; <0.1 % ammonia
- Stability under test conditions: stable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 1.56, 6.25, 25 and 100 mg/L
- Sampling method: At the start of the test (after 0 hours) sampIes from vessels which were not stocked with daphnids were analysed and at the end
of the test (after 48 h) sampIes from stocked and unstocked vessels were analysed.
- Sample storage conditions before analysis: refrigerator
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the preparation of the stock solution (100 mg/L) the test substance was stirred for about 10 min at 22 ± 1 °C in M4-medium.
- Controls: negative control
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: Daphnia magna Straus
- Source: originally obtained from the Institute National de Recherche Chimique Appliquée, France and cultured in the laboratories of the test facility since 1978
- Age at study initiation (mean and range, SD): 2-24 h
- Feeding during test: none
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
2.20 - 3.20 mmol/L
Test temperature:
20.4 - 20.7 °C
pH:
not pH-adjusted: 8.0 - 9.6 (pH > 9 in the two highest test concentrations: 50 mg/L (pH 9.2) and 100 mg/L (pH 9.6))
pH-adjusted: 7.2 - 7.7
Dissolved oxygen:
7.9 - 8.3 mg/L
Nominal and measured concentrations:
Nominal: Control, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 mg/L. Additionally, solutions of 50 and 100 mg/L which were pH adjusted were tested in parallel.
Measured: Recovery after 48 h in test solution stocked with daphnids: 79.9 to 97.2%; unstocked test solutions: 83.3 to 96.4%
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: flat bottom glass tubes, 20 ml nominal volume
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to the test guideline DIN 38412, L 11:
- Ca/mg ratio: 4:1

OTHER TEST CONDITIONS
- Adjustment of pH: yes for two additional concentrations which were tested in parallel (50 and 100 mg/L)
- Photoperiod: 16 : 8 hours day-night regime
- Light intensity: diffuse light (5 microeinstein/m*m*s) at 400 - 750 nm wave length

Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: not pH-adjusted
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: not pH-adjusted and pH-adjusted
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: not pH-adjusted and pH-adjusted
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: pH-adjusted
Details on results:
- Mortality of control: 0%
- Effect concentrations exceeding solubility of substance in test medium: no
- The effect concentrations are based on nominal values since the test concentrations were maintained at ±20% of nominal concentrations except for the lowest test concentration, which was slightly below 80% of nominal.
- The immobilisation was significantly reduced after pH adjustment of the test solutions. In the highest tested concentration (100 mg/L), the immobilisation was reduced to 0% after 48 h (pH 7.2) compared to 45% in the not pH-adjusted test solution (pH 9.6).
Reported statistics and error estimates:
Due to the results of the test a statistical evaluation of the data was unnecessary.

Analytical chemistry

Test concentration

nominal

mean measured

0 h (no daphnids)

48 h (no daphnids)

48 h (stocked with daphnids)

(mg/L)

(mg/L)

(% of nominal)

(mg/L)

(% of nominal)

(mg/L)

(% of nominal)

100

93.9

93.9%

96.0

96.0%

96.1

96.1%

25

23.7

94.8%

24.1

96.4%

24.3

97.2%

6.25

5.9

94.4%

6.0

96.0%

5.9

94.4%

1.56

1.4

89.7%

1.3

83.3%

1.2

76.9%

0

< 1

---

< 1

---

< 1

---

Immobilisation

Nominal conc.(mg/l)

Cumulative immobilisation (n)

3 h

6 h

24 h

48 h

0

0

0

0

0

1.56

0

0

0

0

3.13

0

0

0

0

6.25

0

0

0

0

12.5

0

0

0

0

25

0

0

0

0

50

0

0

0

0

100

0

1

5

9

50*

0

0

0

0

100*

0

0

0

0

*: neutralised sample

pH values

Nominal conc.(mg/l)

pH values after

0 h

48 h

0

8.0

8.0

1.56

8.2

8.0

3.13

8.2

8.0

6.25

8.3

8.0

12.5

8.6

8.1

25

8.9

8.1

50

9.2

8.3

100

9.6

8.7

50*

7.5

7.7

100*

7.2

7.7

*: neutralised sample

Validity criteria fulfilled:
not specified
Conclusions:
The 48h EC50 value of 2-ethoxyethanamine in Daphnia magna was calculated with nominal concentrations and determined to be >100 mg/L. In test solutions in which pH was not adjusted, immobilisation occurred at the highest test concentration (100 mg/L, 9/10 daphnids immobilised after 48 h). The initial pH of the test solution was however outside the recommended range for Daphnia magna (pH = 9.6, recommended range 6-9) and may therefore have effected the observed immobilisation. In test solutions of 50 and 100 mg/L in which pH was adjusted, no mortality was observed at all, indicating that the observed effects are related to the concentration dependent pH increase caused by the test item.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Read across performed with experimental data from the acute toxicity study in Daphnia magna with the related substance 3-isopropoxypropylamine. The results from the acute toxicity study in Daphnia magna with the related substance 2-ethoxyethylamine were included in the read across approach to support the read across justification. The read across justification document is included in IUCLID Section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
65 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Experimental data on 3-isopropoxypropylamine used for read across.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Experimental data on 2-ethoxyethylamine used to support the read across strategy.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
1991-07-24
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The study should be disregarded for several reasons: • No analytical monitoring was performed. • Lots of documentation is missing for a reliable study assessment (e.g., sample purity not mentioned, no information on statistical analysis). • The study was not performed under GLP conditions. • Finally, there were two major limitations which significantly compromised the reliability of the study with regard to both ECHA’s guidance on information requirements and chemical safety assessment, and the OECD TG 202 (2004): • pH adjustment: The pH was not monitored during the study and no pH adjustment was made because this was not requested in ISO standard 6341 (1989). 3-methoxypropylamine is a base. Therefore, one can expect that the pH of the medium was increased by 3-methoxypropylamine and this may have unduly weakened the daphnids. According to OECD TG 202 which is the most widely accepted method and also according to both actualised ISO 6341 (2012) and ECHA’s guidance documents, pH adjustment is necessary in case pH falls below 6 or increases higher than 9 and toxicity is observed. The experiment was not repeated with pH adjustment. • Oxygen level: Oxygen level in the highest treatment after 48 hours was 2.2 mg/L. This was acceptable according to the ISO 6341 (1989) validity criterion (> 2 mg/L), but it is not acceptable according to the OECD TG 202 validity criterion (> 3 mg/L). The criterion is higher in the OECD guideline because it has been shown that the 2 mg/L criterion was not protective enough against adverse effects related to hypoxia (i.e., adverse effects not due to the substance). It should be noted that O2 generally does not fall below 7 mg/L in this kind of studies.
Qualifier:
according to
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Deviations:
not specified
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material: Methoxy-3-propylamine
- Lot/batch No.: 1949/91
Analytical monitoring:
no
Details on sampling:
no data
Vehicle:
not specified
Details on test solutions:
Stock solution at 8.7 g/L in the medium.
Test organisms (species):
Daphnia magna
Details on test organisms:
Age: < 24 hours
Reared in a ASTM medium.
Fed with algae Selenastrum capricornutum, collected in exponential phase of growth.
Test type:
not specified
Water media type:
not specified
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
no data
pH:
no data
Dissolved oxygen:
2.20 mg/L (at the lowest concentration which gives 100% of daphnids immobilised at the end of the study; must be > 2 mg/L)
Salinity:
no data
Nominal and measured concentrations:
no data
Details on test conditions:
Test vessel: closed tubes
Source/preparation of tested concentrations: dilution 1/100, 1/1000
Reference substance (positive control):
yes
Remarks:
Potassium bichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
61.15 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: >23.63 mg/L and < 158.22 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
13.73 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: > 6.34 mg/L and < 29.72 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
< 0.87 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
5 % immobilised in control test (must be < or = 10 %)
Results with reference substance (positive control):
Potassium bichromate: CE50 (24h) = 0.93 mg/L (0.72 mg/L < CE50 (24h) < 1.19 mg/L)
Accepted range: 0.9 - 2.0 mg/L
Validity criteria fulfilled:
no
Conclusions:
The mobility inhibition of Daphnia magna has been investigated through an ISO 6341 guideline study (Arkema, 1991). Daphnia magna were exposed for 48 hours to a solution of methoxy-3-propylamine at a nominal concentration of 8.7 g/L. The EC50 - 24h was determined to be 61.15 mg/L and the EC50 - 48h was 13.73 mg/L.
The NOEC - 48h was < 0.87 mg/L. However this study was disrgarded for the derivation of the PNEC due to major methodological deficiences as listed above.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 May 1998 - 24 July 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The test was not performed in test solutions in which the pH was adjusted to acceptable levels for the test organisms. Therefore, pH may have contributed to the observed effects.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): 3-isopropoxypropylamine
- Substance type: colourless liquid
- Physical state: Liquid
- Analytical purity: 99.8%
- Lot/batch No.: 9708P0073
- Other: Highly soluble at 20°C
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
t=0h: 200, 142.9, 102, 72.9, 52.1, 37.2 mg/L and control
t=48h: 102, 72.8, 52.1, 37.2 mg/L and control
Vehicle:
yes
Details on test solutions:
Ultrapure water is used for preparation of the stock solutions (ultrafiltration, active carbon, ion exchange, filter of 0.22 µm).
The solutions of 3-isopropoxyproylamine in water were prepared on the day the test was performed.
From the substance being soluble at 200 mg/L, a stock solution from 100 mg/L in dilution water was prepared on the day of the preliminary test, and a stock solution of 200 mg/L (first test) or 120 mg/L (second test) on the day of the definitive test.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna Straus, clone 5 or clone A
- Source: not reported, breeding performed at the study laboratory
- Age at study initiation (mean and range, SD): < 24 hours
- Method of breeding: Daphnia are breeded in M7 Elendt medium supplemented with microscopical algae Raphidocelis subcapitata.
- Feeding during test: no

ACCLIMATION
Test animals were not acclimated to test conditions.
Test conditions (temperature, light/dark cycle) similar as during culturing.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
250 mg/L as CaCO3
Test temperature:
20 ± 1 °C
pH:
Values presented are measured during the second definitve test
pH at start: 7.86 (blank control), 8.18 (7.20 mg/L), 8.31 (11.40 mg/L), 8.47 (18.36 mg/L), 8.64 (29.28 mg/L), 8.75 (46.92 mg/L), 9.08 (75 mg/L), 9.42 (120 mg/L)
pH at end: 8.01 (blank control), 8.30 (7.20 mg/L), 8.41 (11.40 mg/L), 8.42 (18.36 mg/L), 8.49 (29.28 mg/L), 8.62 (46.92 mg/L), 8.99 (75 mg/L), 9.65 (120 mg/L)
Dissolved oxygen:
Oxygen concentration at start: not determined
Oxygen concentration at end: 8.0 mg/L (blanck control), 7.5 mg/L (7.20 mg/L), 7.6 mg/L (11.40 mg/L), 7.7 mg/L (18.36 mg/L), 7.9 mg/L (29.28 mg/L), 7.8 mg/L (46.92 mg/L), 7.7 mg/L (75 mg/L), 7.9 mg/L (120 mg/L)
Salinity:
not applicable
Nominal and measured concentrations:
nominal (first definitive test): control + 37.2-52.1-72.9-102.0-142.9-200.0 mg/L
measured (first definitive test - initial): control + 36.9-52.2-72.7-102.1-166.5-204.8 mg/L
measured (first definitive test - final): 37.8-53.05-74.7-103.8 mg/L; blank control, nominal value of 142.9 and 200.0 mg/L are not analysed
nominal (second definitive test): control + 7.2-11.4-18.36-29.28-46.92-75-120 mg/L
The concentrations in the second definitive test were not measured as the concentrations were stable in the first definitive test (nominal concentraitons were equivalent to measured concentrations).
Details on test conditions:
TEST SYSTEM
- Test vessel: Since the substance may be volatile, the test was performed using closed flasks as testing vessels. In order to avoid volatilisation, flasks were entirely filled with test solutions and closed with rubber caps covered with PTFE.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Penicilin flask of 120 mL nominal capacity
- Aeration: not during test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultrapure water (ultrafiltration, active carbon, ion exchange, 0.22 µm filter)
- Ca/mg ratio: 4

OTHER TEST CONDITIONS
Test flasks were incubated in darkness.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.6 (first definitive test), 1.4 (second definitive test)
- Range finding study: nominal concentrations from ranging from 0.1 to 100 mg/L (2 replica per concentration)
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
65 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 46-100 mg/L
Details on results:
Observations at 48 hours (second definitive test):
7.2 mg/L: no immobile daphnids observed
11.4 mg/L: 1 immobile daphnid observed in recipient 1
18.36 mg/L: 1 immobile daphnid observed in recipient 1
29.28 mg/L: no immobile daphnids observed
46.92 mg/L: 1 immobile daphnid observed in recipient 1
75 mg/L: 5 immobile daphnids in recipient 1, 4 immobile daphnids in recipient 2, 3 immobile daphnids in recipient 3, 1 immobile daphnid in recipient 4
120 mg/L: all daphnids were observed to be immobile at this concentration

Remark: the appearance of the solutions was visually checked at the beginning and at the end of the test: solutions were found to be cloudy, coloured.
Results with reference substance (positive control):
The sensitivity is studied every 2 months by a toxicity study with potassium dichromate (determination of the 24h EC50). The results of the last study are generated on 01/07/1998. A 24h-EC50 value of 1.4 mg/L has been observed.
Reported statistics and error estimates:
24h and 48h EC50 were calculated with nominal concentrations by regression analysis using the Probit/log model.

The study was performed in compliance with its quality criteria: immobilisation in the control did not exceed 10% at the end of the test; test daphnids in the control were not trapped at the surface of the water; concentration of dissolved oxygen in the test vessels remained above 2 mg/L at the end of the test and pH did not vary by more than 1 unit; the concentrations of the test substance have been maintained to within 80% of the initial concentration throughout the duration of the test.

Validity criteria fulfilled:
yes
Conclusions:
The 48-EC50 value of 3-isopropoxypropylamine in Daphnia magna was calculated with nominal concentrations by regression analysis using the probit/log model and was observed to be 65 mg/L. It should be noted that the concentration dependent pH increase caused by the test substance may have affected the results, since pH was > 9 in the two highest test concentrations (75 and 120 mg/L), which is outside of the recommended range in the OECD 202 guideline.

Description of key information

The acute toxicity of the source substance for read across 3-isopropoxypropylamine to the water flea, Daphnia magna, was investigated in a 48-hour static test according to the EEC-Guideline 79/831/EEC method C.2 (Arkema, 1999). Based on nominal concentrations, the 48-hour EC50 was estimated to be 65 mg/L. It should be noted that this value should be considered as a worst case value, as the concentration dependent pH increase caused by the test substance may have affected the results of the study (the pH was > 9 in the two highest test concentrations (75 and 120 mg/L), which is outside of the recommended range in the OECD 202 guideline).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
65 mg/L

Additional information

The study on 3-methoxypropylamine from Arkema (1991) received a reliability score of 3 and is considered as a study to be disregarded for endpoint coverage and hazard assessment purposes. This study was performed in accordance with the standard ISO 6341 (1989). The registrant acknowledges that this is an acceptable alternative to the OECD TG 202. However, the study should be disregarded due to two major limitations which significantly compromised the reliability of the study with regards to both ECHA’s guidance on information requirements and chemical safety assessment, and the OECD TG 202 (2004):

* pH adjustment: The pH was not monitored during the study and no pH adjustment was made because this was not requested in ISO standard 6341 (1989). 3-methoxypropylamine is a base. Therefore, one can expect that the pH of the medium was increased by 3-methoxypropylamine and this may have unduly weakened the daphnids. According to OECD TG 202 which is the most widely accepted method and also according to both actualised ISO 6341 (2012) and ECHA’s guidance documents, pH adjustment is necessary in case pH falls below 6 or increases above 9 and toxicity is observed. The experiment was not repeated with pH adjustment. Therefore, it is not possible to distinguish between a potential pH effect and a direct intrinsic toxic effect of the test substance (if any).

*Oxygen level: The oxygen level in the highest treatment after 48 hours was 2.2 mg/L. This was acceptable to the ISO 6341 (1989) validity criterion (> 2 mg/L), but it is not acceptable with regard to the OECD TG 202 validity criterion (> 3 mg/L). The criterion is higher in the guideline because it has been shown that the 2 mg/L criterion was not protective enough against adverse effects related to hypoxia (i.e., adverse effects not due to the substance). It should be noted that O2 generally does not fall below 7 mg/L in this kind of studies.

In addition, there were some other disadvantages of the study that further compromised its reliability:

* No analytical monitoring was performed.

* Lots of documentation is missing for a reliable study assessment (e.g., sample purity not mentioned, no information on statistical analysis).

* The study was not performed under GLP conditions.

For the reasons mentioned above, the registrant concludes that the study of Arkema (1991) is not reliable as the issues above may have impacted the health status of the daphnids and may consequently have overestimated the toxic effects of 3-methoxypropylamine in a way that is not possible to interpret the results of the study in a satisfactory manner. This study should therefore be disregarded and not considered for the derivation of the PNEC nor for classification.

Because the study with 3-methoxypropylamine was disregarded, data on the related substances 2-ethoxyethanamine and isopropoxypropylamine were added to the dossier. The study with 2-ethoxyethanamine yielded a 48-h EC50 value for Daphnia magna of > 100 mg/L, both in test solutions in which pH was adjusted to levels within the recommended range and in test solutions in which pH was not adjusted. However, the results demonstrated that the observed mortality at 100 mg/L in non-adjusted medium was due to the pH increase caused by the test item, as no mortality at all was observed at 100 mg/L in pH-adjusted medium.

In the study with isopropoxypropylamine, a 48-h EC50 of 65 mg/L was obtained for Daphnia magna in test solutions in which pH was not adjusted. No testing was performed in pH-adjusted medium. Since the pH was > 9 in the test solutions with nominal concentrations of 75 and 100 mg/L, the results may have been affected by the increased pH as well. As explained in the read across justification document, the source substance for read across was considered to be isopropoxypropylamine, as a worst case, to cover for the potential existence of direct intrinsic toxic effects of the test substance, which could not entirely be excluded based on the results of the available aquatic toxicity tests. The read across justification document is attached to IUCLID Section 13.