Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
additional ecotoxicological information
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 January to 11 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch number: 2018/019/92
Purity: 98.5% both by UV and autotitrator
Expiry date: 21 January 2020
Analytical monitoring:
yes
Details on sampling:
At the beginning and at the end of the test, the content of the test item in the test solutions was determined using HPLC-UV. The measured concentrations at the start of the test were between 105 % and 107 % of the nominal concentration and between 82 % and 91 % of the nominal concentration at the end of the test. The concentrations 10 mg/L, 22 mg/L and 46 mg/L were diluted before measurement to lie within the calibrated range (10 mg/L dilution factor 4, 22 mg/L and 46 mg/L dilution factor 10). The recovery rate from Daphnia test medium of 108.6 % was also taken into account.
Vehicle:
no
Details on test solutions:
A stock solution containing 100.4 mg/L test item in dilution water was prepared. The concentrations to be tested were prepared by dilution of this stock solution with dilution water.
Test organisms (species):
Daphnia magna
Details on test organisms:
Specification
Species Daphnia magna
Authority STRAUS Strain Berlin
Sex female
Age between 0 and 24 hours
Origin Umweltbundesamt Berlin In-house breeding since 27. September 2007

Selection of the test system was made following the proposal of the guidelines.

Animal Husbandry
Daphnia magna is bred in the LAUS GmbH throughout the year. The animals are kept for the use in toxicity tests. They multiply by parthenogenesis, thus being genetically identical. The keeping is performed similar to the method described in the OECD guideline.
Vessels preserving glasses, nominal volume 2 L
Medium M4-Medium (recipe of ELENDT)
Food green algae (Desmodesmus subspicatus)
Medium renewal twice a week
Photo period 16/8 hours, using neon tubes
Temperature 20  2 °C
Test type:
static
Water media type:
other: Elendt M4
Limit test:
no
Total exposure duration:
48 h
Test temperature:
19.6 - 21.5 oC
pH:
7.5 - 7.7
Dissolved oxygen:
8.0 - 9.0 mg O2 / L
Nominal and measured concentrations:
Time 0: 90.6 - 107 % Nominal
48h: 79.4 - 91% Nominal
See Table below
Details on test conditions:
Selection of Daphnia
23 hours before the start of the test, the adult animals were separated from the young. 0.5 hours before test start, the adults were caught with the help of a glass tube, and the newborn daphnia (age < 24h) were sieved from the medium and immediately placed into a beaker containing dilution water. After the settling-in period, animals which showed no apparent damage were used for the test. Switching from M4-medium (husbandry) to Dilution water (test) has been shown not to cause any detrimental effects for test daphnia, therefore no acclimation period of 48 h was performed. This is a deviation from OECD Guideline No. 202 and EU-Method C.2, which can be considered uncritical.

Study Performance
Using a glass tube, the daphnia were caught and lifted from the beaker. They were put on a small sieve, and the medium surrounding the animals was sucked off using absorbent paper. Immediately after that, the animals were put into the respective test solution. The test vessels were left to stand for 48 hours. After 24 and 48 hours, the immobilised daphnia were counted. Daphnia are considered immobilised when they do not perform any movements or are only able to move their antennae when the beaker is gently agitated. Daphnia which are trapped at the surface of the test solution are also considered immobilised. The pH, the concentration of dissolved oxygen and the content of the test item in the test vessels were measured at the beginning and at the end of the test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
10.39 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study (201902R201). The 24h-EC50 value was determined as 1.98 mg/L. The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as specified in the OECD guideline.
Reported statistics and error estimates:
The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.3.0. No error estimates were provided due to mathematical reasons.

Immobility (cumulative)

 Nominal conc. (mg/L)  24h (%)  48h (%)
 Control  0  0
 2.2  0  0
 4.6  0  0
 10  0  40
 22  50  100
 46  100  100

 

Mean measured Test Item Concentrations

 Nominal conc. (mg/L)  Time 0 (mg/L)  48h (mg/L)  % Nominal (Time 0)  % Nominal (48h)
 Control  0  0  0  0
 2.2  2.31  1.96  105  89
 4.6  4.82  3.93  105  85
 10  10.62  9.12  106  91
 22  23.60  18.07  107  82
 46  48.63  41.88  106  91
         
Validity criteria fulfilled:
yes
Conclusions:
The 48h EC50 value was determined to be 10.39 mg/L.
Executive summary:

The 48h EC50 value was determined to be 10.39 mg/L.

Although the substancei s known to hydrolyse under environmental conditions, analysis showed sufficient stability in the 48 hours of this test under the laboratory conditions used.

The instablity in water is thought to be partly to do with interaction with ions in hard water under alkaline conditions.

Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 May - 31 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch : 2018/019/92
Purity: 98.5% by UV and titrator
Expiry: 21 January 2020
Analytical monitoring:
yes
Details on sampling:
0h and 72h
Vehicle:
no
Details on test solutions:
A stock solution containing 100 mg/L test item in algal medium (demineralised water enriched with minerals but without algae) was prepared. The concentrations to be tested were prepared by dilution of this stock solution with algal medium.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Unicellular freshwater green alga.
Genus Desmodesmus
Species subspicatus
SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae

Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.
Test type:
static
Water media type:
other: OECD TG 201 medium
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.4-24.3oC
pH:
8.3 - 8.5
Details on test conditions:
4440-8880 Lux
Reference substance (positive control):
yes
Remarks:
Potassium dichromate 0.28 and 0.65 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.53 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.53 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.29 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.95 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 18.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
No negative impact on microscopic cell evaluation at all test concentrations.
Results with reference substance (positive control):
72h ErC50 0.65 mg/L
72h EyC50 0.28 mg/L
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software (Microsoft Ex-cel®). The estimation of the biological data was accomplished using the software Tox-Rat® Professional, version 3.3.0. The details of calculation are stated in Annex 4: Statisti-cal calculation using ToxRat® Professional 3.3.0.

Geometric Mean of Measured Concentrations

Nominal
Concentration Test Item

Geometric Mean

mg/L

mg/L

0.32

0.16

1

0.53

3.2

1.79

10

5.46

32

18.62

From the cell numbers, the Growth Rate µ and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Table9.6a      Growth Rate µ, Yield

Nominal Concentration in mg/L

Parameter

Growth Rate

(0-72 h) [day-1]

Yield (0-72 h)
[Cell Concentration/mL]

Blank control

Mean

1.80

529007

SD

0.02

33641

0.1

Mean

1.79

514240

SD

0.01

12341

0.32

Mean

1.79

519620

SD

0.01

8559

1

Mean

1.76

464660

SD

0.01

19577

3.2

Mean

1.50

215007

SD

0.03

19027

10

Mean

1.43

172080

SD

0.00

1273

32

Mean

1.43

170927

SD

0.00

248

SD = Standard Deviation

The following mean inhibition values were calculated for the Growth Rate µ and the Yield.

A clear dose-response relationship could be observed. However, the inhibition in the two highest concentrations was nearly identical. The highest concentration was not used for evaluation so that the graphical evaluation is not falsified.

 Inhibition Values

Nominal concentration in mg/L

% Inhibition

Growth Rate (0-72 h)

Yield (0-72 h)

Blank control

0

0

0.1

0.50

2.79

0.32

0.30

1.77

1

2.37

12.16

3.2

16.57

59.36

10

20.60

67.47

32

20.72

67.69

 

All validity criteria were met

Validity criteria fulfilled:
yes
Conclusions:
ErC50 > 18.62 mg/L
Executive summary:

It is likely that the test material, or degradation products, interfered with the mineral content of the test media causing inhibition of growth due to removal of key nutrients from the small test vessels.  This is something that would not be seen in the wider environment with moving water systems that allow for minerals to be replenished.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: SANCO 3029/99 revision 4
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-azobis[2-methylpropionamidine] dihydrochloride
EC Number:
221-070-0
EC Name:
2,2'-azobis[2-methylpropionamidine] dihydrochloride
Cas Number:
2997-92-4
Molecular formula:
C8H18N6.2ClH
IUPAC Name:
2,2'-azobis[2-methylpropionamidine] dihydrochloride
Test material form:
solid
Specific details on test material used for the study:
Batch; 2018/19/59
Purity: 98.5%
Expiry Date: 7 August 2019

Results and discussion

Any other information on results incl. tables

Specificity

The condition “peak areas in blank < 30 % of the peak areas at the proposed LOQ” was fulfilled.

Calibration

A linear calibration function was calculated. Correlation coefficient  => 0.99 This condition is fulfilled with correlation coefficient 0.99998, the working range 0.05 – 5 mg/L can be used.

Accuracy & Precision

Alagae, Low: Accuracy 103.5%, RSD 4.5%

Algae, High: Accuracy 104.8%, RSD 0.7%

Daphnia, Low: Accuracy 106.8%, RSD 0.9%

Daphnia, High: Accuracy 110.5%, RSD 0.7%

All accuracy values lay in the demanded range of 70 – 110 %, with the exception of the test item solution 46 mg/L in daphnia test medium. However, the deviation of 0.5% is so small that it can be neglected and the repeatability of the measurements of five independent samples is sufficient with an RSD of 0.7 %. As the values lay outside the range 100 ± 5 %, the mean recovery rates will be taken into account in the calculation of the test item concentrations in the subsequent ecotoxicological studies.   The condition for precision “RSD ≤ 20 % per level” was fulfilled for both media and levels.

Stability

The test item was considered unstable in algal test medium under the test conditions as the residual concentration was < 80 % of the initial concentration at the end of the incubation period. Nevertheless, an analytical monitoring of the Algal Growth Inhibition Test is possible. In daphnia test medium under the test conditions was the test item considered stable as the residual concentration was > 80 % of the initial concentration at the end of the incubation period

Applicant's summary and conclusion

Conclusions:
This analytical method is considered valid for the analysis of 2, 2'-azobis[2-methylpropionamidine] dihydrochloride in the matrices Algal and Daphnia test medium.