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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:
2019-03-29 to 2019-05-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 April 2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling: 4 sampling time points (0 h, 24 h (old solution), 24 h (fresh solution) and 48 h) of at each 5 test concentrations + Negative Control (NC) samples, 24 samples prediluted 1:2 with acetonitrile (ACN)
- Substance specific analysis was performed at the test site of menal GmbH.
- The sampling was conducted according to the following specification:
After 0 h exposure: all replicates of NC, A, B, C, D and E were sampled; one sample per replicate; 4 samples of 1 mL per treatment group.
After 24 h exposure (before renewal of test solutions): all replicates of NC, A, B, C, D and E were sampled; one sample per replicate; 4 samples of 1 mL per treatment group.
After 24 h exposure (after renewal of test solutions): all replicates of NC, A, B, C, D and E were sampled; one sample per replicate; 4 samples of 1 mL per treatment group.
After 48 h exposure: all replicates NC, A, B, C, D, E were sampled; one sample per replicate; 4 samples of 1 mL per treatment group.
For each sampled treatment, two of the samples from 0 h, 24 h (old and new test solutions) and 48 h were sent to the analytical laboratory for chemical analysis using an insulated box with thermal packs. The remaining samples were stored as retain samples in the freezer (≤ -18 °C) until finalization of the study.
- Concentrations: NC (nominal 0 mg/L), A (nominal 0.5 mg/L), B (nominal 1.0 mg/L), C (nominal 2.0 mg/L), D (nominal 4.0 mg/L) and E (nominal 8.0 mg/L)
- Sampling method: The samples were filled into 8 mL glass vials. To each vial, 1 mL acetonitrile was added and samples were stored in the freezer (≤ -18 °C).
- Sample storage conditions before analysis: Samples were stored at -20 °C ± 10 °C at menal GmbH until analysis (in medium/ACN, 50/50, v/v) at least until finalization of the phase report after which they will be destroyed or further stored. On assay day, samples were thawed at room temperatures and directly transferred into an HPLC-vial.
- Shipping: Personal handover between Hydrotox GmbH and menal GmbH
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution was prepared by adding 8.1 mg/8.2 mg test item (test start/renewal of test solutions) to 1000 mL test medium and shaking for 24 hours using an overhead shaker at 22.8 - 23.5 °C in the dark. This stock solution was used as highest test concentration in the test. Four replicates, with five daphnids each, were prepared for each test item treatment and the negative control (NC). The further test concentrations were prepared by diluting the stock solution with test medium (dilution factor of 2).
- Controls: The negative control was the test medium.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna Straus
- Origin: The test organism Daphnia magna Straus 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
- Feeding during test: no

CULTURING
The daphnids are cultured at 20 ± 2 °C with 16 h light : 8 h dark and placed into fresh Elendt M4 medium twice a week. They are kept at approx. 10 animals / 200 mL and fed on workdays with Desmodesmus subspicatus algae corresponding to 0.1 mg C × daphnid-1 × day-1. The test organisms used were 2.5 - 22.25 h old at the start of the test.
Quality assurance takes place at regular intervals by testing the sensitivity of the test organisms to the reference item potassium dichromate (Honeywell Speciality Chemicals Seelze GmbH, Germany, Lot no.: H1030, expiration date: Sept. 2022). The recent quality testing was performed in November 2018 with EC50 (24h) = 1.45 mg/L (CL 95 %: 1.34–1.57 mg/L), which is in the declared range of 0.6–2.1 mg/L (24 h) according to OECD 202.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
Evaluation after 24 and 48 hours
Hardness:
Not reported
Test temperature:
20.4 - 21.5 °C
pH:
The pH was 7.8 - 8.1 in the control and 7.9 - 8.1 in the test item treatments
Dissolved oxygen:
8.0 - 8.3 mg/L in the control and 8.0 - 8.02 mg/L in the test item treatments
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal test item concentrations [mg/L]: 0.5, 1.0, 2.0, 4.0 and 8.0
Measured test item concentrations (geometric mean) [mg/L]: 0.11 (for measurements
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers 50 mL
- Type of flow-through (e.g. peristaltic or proportional diluter): semi-static, no flow-through
- 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: Reconstituted Water (Elendt "M4"), according to OECD 202
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH as well as the oxygen concentrations were measured at the start (0 h), before and after renewal of the test solutions (24 h) and at the end of the test (48 h). The temperature was measured continuously.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Lowest and no observed effect concentrations of test item on immobility and mortality

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
A preliminary range finding test without GLP was performed before the start of this GLP-study.
- Test concentrations (in range finding study): Nominal concentrations of 0.1, 1.0 and 10 mg/L test item were tested and resulted in 0%, 5% and 100% immobility (48 h), respectively.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.83 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
3.88 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
1.45 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
1.72 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
2.06 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
LOEC
Effect conc.:
5.44 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.57 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
2.06 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: Besides immobility no abnormalities are reported
- Mortality of control: no mortality
- Other adverse effects control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Potential degradation product of the test item was observed during the pre-tests and also taken into account during the chemical analysis by manual integration of both, the peak of the test item as well as a second peak (presenting the degradation product) directly eluting after the test item (semi-quantitative approach).
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.
Depending on the sensitivity of the available analytical method at the test site, the concentrations detected were below the LOQ (= 0.1 mg/L). Some concentrations were determined above the LOD (= 0.05 mg/L) and the LOQ. Those values were considered for determination of ECx values. For nominal loadings within which the concentrations were below the LOD, the LOD was used for calculation of the geometric mean.

As the measured test item concentrations are not within ± 20% of the nominal concentrations, according to OECD 202 (2004), all results are given in relation to the analytically measured test item concentrations (geometric mean concentrations).

During the pre-tests for this study, an unknown potential degradation product (probably an oxidation product of the test item) with a similar mass as the test item was observed as a second peak in the chromatogram diluting directly after the test item. This potential degradation product was not identified until this study was conducted. As the pre-test data showed that integrating both peaks resulted in a recovery in the range 80 - 120%, this second peak was taken into account during the chemical analysis for the present study by manual integration of both, the peak of the test item as well as a second peak presenting the potential degradation product.

The immobilities of the test organisms in the control (NC) and the test item treatments are summarized in Table 1.

Table 1: Immobility after 24 h and 48 h exposure

Nominal test conc. [mg/L]

Daphnia introduced

Immobile 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

5

5

5

5

0

0

0

0

0

0

0

0

0

0

0.5

5

5

5

5

0

0

0

0

0

0

0

0

0

0

1.0

5

5

5

5

0

0

0

0

0

0

0

0

0

0

2.0

5

5

5

5

0

0

0

0

0

0

0

0

0

0

4.0

5

5

5

5

0

0

0

0

0

1

0

1

0

10

8.0

5

5

5

5

4

3

5

4

5

5

5

5

80

100

pH and oxygen concentrations after 0 h, 24 h (before and after renewal) and 48 h are shown in Table 2.

Table 2: pH and oxygen concentrations

Nominal test conc. [mg/L]

pH

Oxygen concentration [mg/L]

0 h

24 h (old)

24 h (new)

48 h

0 h

24 h (old)

24 h (new)

48 h

NC

8.1

8.0

7.8

7.9

8.2

8.3

8.0

8.1

0.5

8.0

8.0

7.9

7.9

8.0

8.1

8.0

8.1

1.0

8.1

8.0

7.9

7.9

8.0

8.1

8.0

8.1

2.0

8.1

8.0

7.9

8.0

8.1

8.1

8.1

8.1

4.0

8.0

8.0

7.9

7.9

8.1

8.0

8.1

8.2

8.0

8.0

7.9

7.9

7.9

8.2

8.1

8.1

8.1

Analytically measured test item concentrations after 0 h, 24 h and 48 h exposure are illustrated in Table 3.

Table 3: Analytically measured test item concentrations

Nominal test item concentration [mg/L]

Sampling time [h]

Measured test item concentration [mg/L]

Recovery [%nominal]

Geometric mean test item concentration [mg/L]

NC

0

< LOD

-

-

24 (old)

< LOD

-

24 (new)

< LOD

-

48

< LOD

-

0.5

0

0.26

52.0

0.11*

24 (old)

< LOD

-

24 (new)

0.24

48.0

48

< LOD

-

1.0

0

0.55

55.0

0.17*

24 (old)

< LOD

-

24 (new)

0.57

57.0

48

< LOD

-

2.0

0

1.15

57.5

0.57

24 (old)

0.31

15.5

24 (new)

1.49

74.5

48

0.20

10.0

4.0

0

2.35

58.8

2.06

24 (old)

1.63

40.8

24 (new)

3.09

77.3

48

1.53

38.3

8.0

0

4.82

60.3

5.44

24 (old)

6.48

81.0

24 (new)

5.91

73.9

48

4.75

59.3

*For measurements which are < LOD, the LOD was used for calculation of the geometric mean

Analytically measured concentrations of the test item and the potential degradation product (sum of both peaks) are given in Table 4.

Table 4: Analytically measured concentrations of the test item and the potential degradation product (sum of both peaks)

Nominal test item concentration [mg/L]

Sampling time [h]

Measured test item concentration [mg/L]

Recovery [%nominal]

Geometric mean test item concentration [mg/L]

0.5

24 (new)

0.26

52.0

0.26

1.0

0

0.58

58.0

0.62

24 (new)

0.66

66.0

2.0

0

1.30

65.0

0.64

24 (old)

0.33

16.5

24 (new)

1.78

89.0

48

0.22

11.0

4.0

0

2.68

67.9

2.51

24 (old)

1.92

48.0

24 (new)

3.85

96.3

48

2.00

50.0

8.0

0

5.62

70.3

7.24

24 (old)

9.29

116.1

24 (new)

7.44

93.0

48

7.07

88.4

CRITERIA OF VALIDITY

- Immobilisation of the test organisms in the control at the end of the test was 0% and therefore 10%.

- Dissolved oxygen concentration in the control and the test item treatments at the end of the test was ≥ 8.0 mg/L and therefore ≥ 3 mg/L.

- The test is valid according to OECD Test Guideline 202 (April 2004).

Validity criteria fulfilled:
yes
Conclusions:
In an acute immobilisation test according to OECD guideline 202, Daphnia magna were exposed to the test item for 48 h and the following effect concentrations were determined:
48-h EC50 = 2.83 mg/L [95 % C.I.: 2.30 to 3.55 mg/L]
48-h EC10 = 1.45 mg/L [95 % C.I.: 1.05 to 1.82 mg/L]
48-h NOEC = 0.57 mg/L
48-h LOEC = 2.06 mg/L
Executive summary:

The 48-hr-acute toxicity of 1,3,5-Tris-(3-mercaptopropyl)isocyanurate to Daphnia magna was studied under semi-static conditions. Test species were exposed to control, and test chemical at nominal concentrations of 0.5, 1.0, 2.0, 4.0, and 8.0 mg/L, corresponding to measured concentration of 0.11, 0.17, 0.57, 2.06, and 5.44 mg/L for 48 h. Immobilisation was observed daily.  The 48-hour EC50 was 2.83 mg/L.  The 48-hour NOEC based on immobilisation was 0.57 mg/L.

 

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. 1stinstar): < 24 h

Test Type (Flowthrough, Static, Static Renewal): Semi-static

 

48-h EC50 = 2.83 mg/L [95 % C.I.: 2.30 to 3.55 mg/L]

48-h EC10 = 1.45 mg/L [95 % C.I.: 1.05 to 1.82 mg/L]

48-h NOEC = 0.57 mg/L

48-h LOEC = 2.06 mg/L

Endpoint(s) Effected: Immobilisation

Description of key information

In an acute immobilization test according to OECD guideline 202, Daphnia magna were exposed to the test item for 48 h and the following effect concentrations were determined:

48-h EC50 = 2.83 mg/L [95 % C.I.: 2.30 to 3.55 mg/L]

48-h EC10 = 1.45 mg/L [95 % C.I.: 1.05 to 1.82 mg/L]

48-h NOEC = 0.57 mg/L

48-h LOEC = 2.06 mg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
2.83 mg/L

Additional information