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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-30 to 2018-03-28
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
- Sampling method: The samples were filled into 10 mL glass vials. To each vial, 5 mL acetonitrile was added and samples were stored in the freezer (≤ -18 °C). The sampling was conducted according to the following specification:
• After 0 h, 24 h and 48 h exposure, all replicates of NC (= 0), A (= 25 mg/L), B (= 50 mg/L), C (= 100 mg/L), D (= 200 mg/L) and E (= 400 mg/L) were sampled; one sample per replicate: 4 samples of 5 mL per treatment group.
For each sampled treatment, one of the samples from 0 h, 24 h and 48 h was sent to the analytical laboratory at the test site menal GmbH for chemical analysis.
- Concentrations: NC, A, B, C, D and E
- Shipment: Personal handover between Hydrotox GmbH and menal GmbH
- Sample storage conditions before analysis: All samples were stored in a freezer at ≤ - 18 °C ± 10 °C until analysis
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution was prepared as Water Soluble Fractions (WSF) by adding 400.1 mg test item to 500 mL test medium and shaking for 48 h using an overhead shaker at 21.4 – 23.0 °C in the dark. The WSF was filtered through a fibre-glass filter with a retaining range up to 0.6 µm (MN 85/70 BF, Marchery-Nagel, Düren, Germany). The filter was prepared by rinsing with purified water and preconditioning with ca. 100 mL WSF (which was discarded) to reduce adsorption of the test item.
- Differential loading: The further test loading rates were prepared by diluting the stock solution with test medium.
- Controls: The negative control (NC; test medium) was treated in the same way as the test item solution.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Waterflea
- Scientific name: Daphnia magna Straus
- Source: 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

- Acclimation period: no – culturing conditions same as test: 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/day. The test organisms used were 1.0 - 22.5 h old at the start of the test.

- Quality assurance: Quality assurance takes place at regular intervals by testing the sensitivity of the test organisms to the reference item potassium dichromate (Sigma, Steinheim, Germany; Lot no.: MKBF2111V, expiration date: January 2018). The recent quality testing was performed in August 2017 with EC50 (24h) = 1.77 mg/L (CL 95 %: 1.63 – 2.05 mg/L), which is in the declared range of 0.6 – 2.1 mg/L (24 h) according to OECD 202.
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
not reported
Test temperature:
19.5 - 21.0 °C
7.6 - 7.7 (control); 7.6 - 7.8 (test item)
Dissolved oxygen:
8.2 - 8.4 mg/L (control); 8.1 - 8.4 mg/L (test item)
not applicable
not reported
Nominal and measured concentrations:
25, 50, 100, 200, 400 mg test item/L (nominal) and control
All measured test item concentrations (geometric mean): 0.98, 1.98, 3.51, 5.35, 8.87 mg/L
Details on test conditions:
- Test vessel: glass beakers 50 mL
- Type of flow-through (e.g. peristaltic or proportional diluter): static, no flow-through
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

- 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 water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.

- 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


Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions. The pre-experiments were not performed in compliance with the GLP-Regulations and were excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the project number of the present study.
- Test concentrations: Nominal loading rates of 10, 100 and 200 mg/L test item were tested and resulted in 10, 0 and 10 % immobility (48 h), respectively.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
Potassium dichromate
48 h
Dose descriptor:
Effect conc.:
8.291 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CL: 7.284 - 10.004
48 h
Dose descriptor:
Effect conc.:
5.913 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CL: 4.114 - 6.819
48 h
Dose descriptor:
Effect conc.:
8.87 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
48 h
Dose descriptor:
Effect conc.:
5.35 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
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.


In the control, the test item was not detected. The measured test item concentrations in the test item treatments were < LOQ - 4.56 % of the nominal loading rates. As the measured test item concentrations are not within ± 20 % of the nominal loading rates, according to OECD 202 (2004) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations. Therefore, the geometric mean of all three measurement times (0 h, 24 h and 48 h) was calculated (see Table 1).

Table 1. Analytically measured test item concentrations after 0 h, 24, h, and 48 h of exposure

 Nominal test item loading rate (mg/L)  Sample  Sampling time (h)  Measured test item concentration (mg/L)  Geometric mean test item concentration (mg/L)
 NC  NC1  0  <LOQ  --
 NC  NC2  24  <LOQ  --
 NC  NC3  48  <LOQ  --
 25  A1  0  <LOQ (0.824)  0.98
 25  A2  24  1.11  0.98
 25  A3  48  1.04  0.98
 50  B1  0  1.53  1.98
 50  B2  24  2.28  1.98
 50  B3  48  2.23  1.98
 100  C1  0  2.97  3.51
 100  C2  24  3.70  3.51
 100  C3  48  3.95  3.51
 200  D1  0  6.18  5.35
 200  D2  24  5.01  5.35
 200  D3  48  4.95  5.35
 400  E1  0  16.50  8.87
 400  E2  24 6.38   8.87
 400  E3  48  6.38  8.87

- BIOLOGICAL RESULTS: 60% of test organisms were immobilised at nominal test loading rate of 400 mg/L (see Table 2).

Table 2. Immobility after 24 h and 48 h exposure

 Nominal test item loading rate (mg/L)  Geometric mean test item concentration (mg/L)  Immobility, 24 h (%)  Immobility, 48 h (%)
 NC  --  0  0
 25  0.98  0  0
 50  1.98  0  0
 100  3.51  0  0
 200  5.35  0  5
 400  8.87  0  60


-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 ≥ 8.1 mg/L and therefore ≥ 3 mg/L.

Therefore, the test is valid according to OECD Test Guideline 202 (13 April 2004).

Validity criteria fulfilled:
In an acute toxicity test according to the OECD guideline 202 with Daphnia magna an EC50 of 8.291 mg/L (meas., geom. mean) was determined for N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine.
Executive summary:

The 48-hr-acute toxicity of N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine to Daphnia magna was studied under static conditions. Daphnids were exposed to control, and test chemical at measured concentrations of 0,0.98, 1.98, 3.51, 5.35 and 8.87 mg/L (nominal: 0, 25, 50, 100, 200, and 400 mg/L) for 24 and 48 hours. Mortality, immobilization and sublethal effects were observed daily. The 48– hour EC50 was 8.291 mg a.i./L. The 48-hr NOEC based on immobilization equals 5.35 mg/L.

The initial concentration and the maintenance of the exposure concentrations during the test were verified in the analytical part. As the measured test item concentrations are not within ± 20 % of the nominal loading rate, according to OECD 202 (2004) and OECD 23 (2000), all results are given in relation to the analytically measured test item concentrations.

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): < 24 h

Test Type (Flowthrough, Static, Static Renewal): Static


NOEC: 5.35 mg a.i./L (based on LOQ)

LOEC: 8.87 mg a.i./L

EC50: 8.291 mg a.i./L ( CL 95 % = 7.284 – 10.004)


Endpoint(s) Effected: mobility, mortality

Description of key information

The toxicity of N2 -Isobutyryl-5'-O-(4,4'-Dimethoxytrityl)-2'-deoxyguanosine was assessed in a 48 -hour acute toxicity test according to OECD test guideline 202 with Daphnia magna.

NOEC: 5.35 mg a.i./L (based on LOQ)

LOEC: 8.87mg a.i./L

EC50: 8.291 mg a.i./L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
8.291 mg/L

Additional information