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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:
2017-03-03 to 2017-06-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 201601004
- Expiration date of the lot/batch: 01.01.2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in dark, cool, dry place in tightly closed receptacles
- Stability under test conditions: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The stock solutions (one for the start and one for the renewal of the test solutions at 24 h) were prepared by adding 64.3 mg (0 h) and 64.1 mg/L (24 h) test item to 1000 mL test medium and
shaking for 2.5 h using an overhead shaker at 22.3 – 22.4 °C in the dark, respectively. This stock solution was used as highest test item concentration in the test.
Analytical monitoring:
yes
Details on sampling:
- nominal concentrations (mg/L): 0.0 (sampling code: NC); 4.0 (sampling code: A); 8.0 (sampling code: B); 16.0 (sampling code: C); 32.0 (sampling code: D); 64.0 (sampling code: E)
- Sampling method:
The samples were filled into 10 mL glass vials and measured at the same day (0 h and 24 h exposure) or stored at ≤ -18 °C until measuring at the following day (48 h exposure). The sampling was conducted according to the following specification:
• At the start of the test (0 h), the test solutions NC, A, B, C, D and E were sampled before division to the test vessels (each group: 4 samples of 6 mL).
• After 24 h exposure:
• Before renewal of the test solutions, all replicates of NC, A, B, C, D and E were sampled (one sample per replicate: 4 samples of 6 mL per group).
• The new test solutions NC, A, B, C, D and E were sampled before division to the test vessels (each group: 4 samples of 6 mL).
• At the end of the test (48 h), all replicates from NC, A, B, C, D and E were sampled (one sample per replicate: 4 samples of 6 mL per group).

Of each sampled treatment, one of the samples from 0 h, 24 h before renewal, 24 h new test solutions and 48 h was sent to the analytical laboratory at the test site menal GmbH and analysed.
The remaining samples were stored as retain samples at ≤ -18 °C until finalization of the study.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: stock solutions were prepared by adding test item (64.3 mg (0h) and 64.1 mg/L (24 h)) to test medium (1000 mL) and shaked for 2.5 h using an overhead shaker at 22.3 – 22.4°C in the dark, respectively.
- Controls: yes
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): no vehicle


- Range finding test: yes
- A preliminary test without GLP was performed before start of this GLP-study.
Nominal concentrations of 10, 30 and 90 mg/L test item resulted in 15, 80, and 100 % immobility after 48 h exposure, respectively.
Therefore nominal concentrations of 4 – 64 mg/L test item were set in the presented study.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna Straus
- Source: originates from the clone breeding of the Federal Environment Agency, department IV 2.4, Berlin, and has been cultivated at Hydrotox GmbH since October 2012.
- Age of parental stock (mean and range, SD): unknown
- Age at study initiation: 0.5 – 20.25 h old
- Feeding during test: Not reported
- Food type during breeding: Desmodesmus subspicatus algae
- Amount and frequency during breeding: Daily (on workdays) 0.1 mg C per daphnid and per day

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20.6 - 21.3°C
pH:
pH of the test item treatment: 7.4 - 7.7
pH of the control: 7.7
see also table 1 "pH and O2 -concentrations after 0 h, 24 h (before and after renewal) and 48 h exposure" in "Any other information on materials and methods incl. tables" below
Dissolved oxygen:
in test item treatment: 7.9 - 8.1 mg/L
in control: 7.6 - 8.2 mg/L
see also table 1 "pH and O2 -concentrations after 0 h, 24 h (before and after renewal) and 48 h exposure" in "Any other information on materials and methods incl. tables" below
Conductivity:
686 μS/cm / 677 μS/cm
Nominal and measured concentrations:
Concentrations of the test item (nominal): 0.0; 4.0; 8.0; 16.0; 32.0; 64.0 mg/L
Concentrations of the test item (measured): 0.0; 1.44; 2.37; 3.75; 15.86; 45.21 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Material, size, fill volume: 50 mL
- Aeration: Yes: After addition of the stock solutions, except the vitamin stock solution S23, the medium (before renewal / after renewal) was aerated for 3.0 h (0h) and 2.5 h (24 h) to achieve oxygen saturation.
- Type of flow-through (e.g. peristaltic or proportional diluter): No: Semi-static system.
- Renewal rate of test solution (frequency/flow rate): Yes, after 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM
The test was performed with M4 medium according to OECD 202 (2004).

OTHER TEST CONDITIONS
- Adjustment of pH: 7.4 - 7.7
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mobility, Mortality

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Spacing factor test concentration: 3
- Test concentrations: 10, 30, 90 mg/L
- Results used to determine the conditions for the definitive study: Nominal concentrations of 10, 30 and 90 mg/L test item resulted in 15, 80 and 100 % immobility after 48 h exposure, respectively. In the present study, nominal concentrations of 4 – 64 mg/L test item were applied.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
56.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
other: EC20
Effect conc.:
47.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
42.92 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
LOEC
Effect conc.:
> 45.21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
>= 45.21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
19.06 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
other: EC20
Effect conc.:
11.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
8.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
15.84 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
3.75 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: not reported
- Mortality of control: no
- Other adverse effects control: not reported
- Abnormal responses: not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
See also table 2 " Immobility after 24 h and 48 h exposure" in "Any other information on results" incl. tables" below
Results with reference substance (positive control):
- Results with reference substance valid?: yes

Reported statistics and error estimates:
As the measured concentrations are not within ± 20 % of the nominal concentrations, according to OECD 202 (2004) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations.
The effect concentrations after exposure were calculated with the statistical software ToxRat Professional 3.2.1.

Table 3: Immobility after 24 h and 48 h exposure.

Nominal test item concentration [mg/L] Geometric mean test item concentration [mg/L] Mobile Daphnia (replicates 1 - 4) Immobility [%]
0 h 24 h 48 h 24 h 48 h
1 2 3 4 1 2 3 4 1 2 3 4
NC NC 5 5 5 5 5 5 5 5 5 5 5 5 0 0
4 1.44 5 5 5 5 5 5 5 5 5 5 5 5 0 0
8 2.37 5 5 5 5 5 5 5 5 5 5 5 5 0 0
16 3.75 5 5 5 5 5 5 5 5 5 5 5 5 0 5
32 15.86 5 5 5 5 5 5 5 5 4 4 4 4 0 20
64 45.21 5 5 5 5 5 4 5 3 0 0 0 0 15 100
Validity criteria fulfilled:
yes
Conclusions:
For the conducted semi-static acute immobilization test with Daphnia magna, applying nominal concentrations of 0 to 64 mg/L propyl gallate, the following effect levels were calculated:
- 48 hr EC50 (measured): 19.06 mg/L (95%-CI: n.d.);
- 48 hr EC20 (measured): 11.15 mg/L (95%-CI: n.d.);
- 48 hr EC10 (measured): 8.43 mg/L (95%-CI: n.d.);
- 48 hr LOEC: 15.84 mg/L;
- 48 hr NOEC: 3.75 mg/L.
Executive summary:

The 48–hr-acute toxicity of propyl gallate to Daphnia magna Straus was studied under semi-static conditions. Daphnids were exposed to a control containing no test substance, and propyl gallate at nominal concentration of 4.0, 8.0, 16.0, 32.0 and 64.0 mg/L (measured concentrations of 0.00 (control), 1.44, 2.37, 3.75, 15.86 and 45.21 mg/L (geometric mean) for 48 hr.

Immobilization was observed daily. 

The 48 – hr EC50 was 19.06 mg/L. 

The 48 – hr NOEC based on immobilization was 3.75 mg/L.

Based on the results of this study, propyl gallate would not be classified as toxic to Daphnia magna Straus in accordance with the CLP classification system.  

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.  

Results Synopsis

Test Organism Age (e.g. 1st instar): 0.5 - 20.25 h

Test Type: Static Renewal

              

- 48 hr EC50: 19.06 mg/L; 95% C.I.: not determined

- 48 hr EC20: 11.15 mg/L; 95% C.I.: not determined

- 48 hr EC10:  8.43 mg/L; 95% C.I.: not determined

- 48 hr LOEC: 15.84 mg/L and

- 48 hr NOEC:  3.75 mg/L.

Description of key information

The lowest valid 48-hr EC50 is determined to be 19.06 mg/L by Schwartz (2017) in a GLP guideline study conforming to OECD 202.

This toxicity study is classified as acceptable and satisfies the guideline requirements for an acute immobilisation test conducted with Daphnia magna.

Furthermore, one publication reported by Zurita et al. (2007) is available, where the effect of propyl gallate to D. magna was determined in a 48-hour acute toxicity test according to OECD guideline 202.

The 48-hr EC50 was determined to be 37.77 mg/L (nominal concentration). The result of this study demonstrated that the crustacean D. magna was very sensitive to propyl gallate.

Since detailed information on the test item or study system are missing and only nominal concentrations are used although the test item is susceptible to hydrolysis, this study is regarded methodologically deficient hence not suitable for the environmental hazard assessment of propyl gallate. Consequently, this toxicity study is disregarded.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
19.06 mg/L

Additional information