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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-03-24 to 1999-07-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
Part I; Adopted April 4, 1984
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
December, 1992
Qualifier:
according to guideline
Guideline:
other: ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus - Acute toxicity test
Version / remarks:
Third edition, 1996-04-01
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(3,5-Dihydroxyphenyl)-1-oxo-2-(N-1-(4-methoxyphenyl)isopropylamino)ethane Hydrobromide
EC Number:
928-779-0
Molecular formula:
C18-H21-N-O4 x HBr
IUPAC Name:
1-(3,5-Dihydroxyphenyl)-1-oxo-2-(N-1-(4-methoxyphenyl)isopropylamino)ethane Hydrobromide
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
During the final test:
- Concentrations: 0.22, 1.0 and 10 mg/L and control at t=0 and t=48
- Sampling method: 10 ml from the approximate centre of the test vessel
- Sample storage conditions before analysis: The samples not analysed on the day of sampling were stored in a deep freeze

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Preparation of test solutions started with stock solutions at 10 or 100 mg/L in ISO-medium. The stock solutions were dissolved applying magnetic stirring and, when necessary, a short period of ultrasonic treatment. The lower test concentrations were prepared by subsequent dilutions of the stock solutions. Test solutions were left standing for approximately 24 hours in the final study and the range-finding test when testing the toxicity of the degraded product. All final test solutions were clear and colourless. The 100 mg/L solution became slightly yellow after 24 hours of standing.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia Magna
- Strain: Daphnia Magna (Crustacea, Cladocera) (Strauß, 1820)
- Age at study initiation (mean and range, SD): young daphnia with an age of < 24 hours
- Method of breeding (culture): Maximum age of the cultures: 4 weeks; Renewal of the cultures: After 7 days of cultivation half of the medium twice a week; Type and amount of food: Daily, a suspension of fresh water algae; Medium: Elendt M7
- Feeding during test: no







Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Test temperature:
measured daily in one blank control: 20.6 - 21.2°C

pH:
t=0h: 7.7-8.0
t=48h: 8.0-8.2
Dissolved oxygen:
t=0h: 9.0 - 9.1
t=48h: 8.3 - 8.5
Salinity:
not applicable
Nominal and measured concentrations:
see table 1 to 4
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all glass
- Aeration: No aeration of the test solutions
- Medium: ISO, prepared in milli-RO water
- No. of organisms per vessel: 10 per vessel containing 80 ml medium
- No. of vessels per concentration (replicates): 2
- - No. of vessels per control (replicates): 2
- Introduction of daphnia: 23 hours after preparation of the test solutions


TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: yes

OTHER TEST CONDITIONS
- Photoperiod: 16 hours daily

TEST CONCENTRATIONS
- Range finding study: combined range finding/limit test: control, 0.1, 1.0, 10, 100 mg/l; second and third range finding test: 0.1, 1.0, 10, 100 mg/l
- Test concentrations: control, 0.22, 0.46, 1.0, 2.2, 4.6, 10 mg/l
- Results used to determine the conditions for the definitive study: Based on the result of the range finding tests, the degradation product(s) prepared at TH 1165 A Keton 1 concentrations from 0.22 to 10 mg/l, increasing by a factor of 2.2
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
other: pre-incubation of the test solutions for 24 hours to ensure testing of the degradation products at test item concentrations.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: pre-incubation of the test solutions for 24 hours to ensure testing of the degradation products at test item concentrations
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.9 mg/L
Nominal / measured:
nominal
Conc. based on:
other: pre-incubation of the test solutions for 24 hours to ensure testing of the degradation products at test item concentrations.
Basis for effect:
mobility
Remarks on result:
other: 95% 2.5-3.4 mg/l
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All final test solutions were clear and colourless. The 100 mg/l solution became slightly yellow after 24 hours of standing.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Mortality: no data about mortality, immobility was measured
- EC50/LC50: 24h-EC50 = 1.1 mg/l (95% CL 0.93 - 1.4 mg/L) and 48h-EC50 = 0.84 mg/l (95% CL 0.76 - 1.0 mg/L)
Reported statistics and error estimates:
Calculation of EC50:
The EC50-value was calculated at 48 hours of exposure from the probits of the percentages of affected daphnia and the logarithms of the corresponding test substance concentrations using the maximum likelihood estimation method (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).

Any other information on results incl. tables

Analytical results:

Table 1: Concentrations of the test item in test medium (combined range-finding/limit test)

      Concentration       
 Time of sampling (hours) Date of sampling  Date of analysis   Nominal (mg/l) Analysed*  Relative to nominal (%) 
 0 24 -03 -99  26 -03 -99**  n.d. n.a. 
 0 24 -03 -99   26 -03 -99**  100  70  70 
 48 26 -03 -99    26 -03 -99   0 n.d.  n.a. 
 48  26 -03 -99   26 -03 -99    100 n.d.   -

Table 2: Concentration of the test item in test medium (extra test)

      Concentration       
 Time of sampling (hours) Date of sampling  Date of analysis   Nominal (mg/l) Analysed*  Relative to nominal (%) 
 0 26 -04 -99  26 -04 -99 1 0.775 78 
 0 26 -04 -99   26 -04 -99  10 8.40 84 
0  26 -04 -99    26 -04 -99   100 108  108 
 24  27 -04 -99   27 -04 -99   1 n.d.   -
 24  27 -04 -99   27 -04 -99     10  n.d.  -
24   27 -04 -99   27 -04 -99    100  23.8  24
48   28 -04 -99   28 -04 -99**    1  n.d.  -
48     28 -04 -99  28 -04 -99**   10  n.d.  -
 48    28 -04 -99   28 -04 -99**   100  0.225***  0.2
 0  26 -04 -99     28 -04 -99**   1  0.554  54
 0  26 -04 -99     28 -04 -99**   10  7.80  78
 0   26 -04 -99    28 -04 -99**   100  119  119

The limit of detection (S/N=3) was determined to be 0.04 mg/l at an injection volume of 10µl (i.e. 0.4 ng absolute). Taking into account the dilution factor of the sample, the concentration of the test item in the 1 and 10 mg/l nominal samples (24 and 48 hour) has to be < 0.08 mg/l.

Stability during storage:

Extra samples were taken at t=0 h and frozen for 2 days to examine the stability of the test item in test medium during storage. For the 1, 10 and 100 mg/l nominal samples, the relative to nominal concentrations were 78, 84 and 108 mg/l nominal samples respectively when analysed freshly and 54, 78 and 119% respectively when analysed after storage. From the results it was concluded that the test item might be unstable in test medium during a storage in a deep freeze for 2 days.

Table 3 and 4 show the analytical results of the final test.

Calibration solutions of the hydrolysis product of the test item were prepared at NOTOX B.V. from standard solutions of the test item in ISO-medium. Because the chemical structure of the hydrolysis product was not known, it was not possible to express concentrations analysed as mg/l hydrolysis product. Concentrations are therefore expressed as the equivalent test item concentration.

No test substance (test item) was detected in the samples from the final test.

Table 3: Concentrations of the hydrolysis product (peak 1) in test medium (final test)

      Concentration****       
 Time of sampling (hours) Date of sampling  Date of analysis   Nominal (mg/l) Analysed*  Relative to nominal (%) 
 0 30 -06 -99   30 -06 -99 ***** n.a. 
 0  30 -06 -99    30 -06 -99 0.22 0.163 74 
0   30 -06 -99     30 -06 -99    1 1.35  135 
0    30 -06 -99      30 -06 -99 10 12.8   128
 48   02 -07 -99    02 -07 -99     0  0.270***  n.a.
48    02 -07 -99    02 -07 -99   0.22  0.665  302
 48     02 -07 -99     02 -07 -99  1  1.69 169 
 48     02 -07 -99     02 -07 -99  10***  14.0***  140

Table 4: Concentrations of the hydrolysis product (peak 2) in test medium (final test)

      Concentration****       
 Time of sampling (hours) Date of sampling  Date of analysis   Nominal (mg/l) Analysed*  Relative to nominal (%) 
 0 30 -06 -99   30 -06 -99 n.d. n.a. 
 0  30 -06 -99    30 -06 -99 0.22  n.d.
0   30 -06 -99     30 -06 -99    1  n.d. -
0    30 -06 -99      30 -06 -99 10 0.900  9
 48   02 -07 -99    02 -07 -99     0   n.d.  n.a.
48    02 -07 -99    02 -07 -99   0.22   n.d.  -
 48     02 -07 -99     02 -07 -99  1   n.d.
 48     02 -07 -99     02 -07 -99  10  1.08  11

* Mean of duplicate analysis. The maximum deviation between the responses was calculated for each sample and was <10%

** Samples were frozen until analysis

*** Extrapolated from the regression line

**** Concentrations are not concentrations of the hydrolysis product but concentrations of the equivalent test item concentration

***** A small peak was detected with the same retention time as peak 1

n.d. not detected

n.a. not applicable

Results Final Test:

Analysis indicated that during the 24-hour incubation period all test substance was converted in its degradation products.

Recoveries based on peak 1 in the samples taken from the degradation product of the test item prepared a nominally 1.0 and 10 mg/l remained fairly stable between 128 and 169% during the 48 hour exposure period. The recovery in the sample taken from 0.22 mg/l increased from 74 to 302% of nominal.

Analysis based on peak 2 showed that measured concentrations were below the limit of detection at nominal 0.22 and 1.0 mg/l. The concentration measured at nominal 10 mg/l remained stable at approximately 1 mg test item equivalents per litre.

The results of analysis should be interpreted with care, as the analytical method used was not a specific method and ws not validated. Hence, it can be concluded that testing involved the degradation products of test item only.

Immobility:

The responses recorded in this test allowed for reliable determination of an EC50. The responses recorded were in agreement with what was expected based on the results of the range finding tests.

Table 5: Acute immobilisation of daphnia after 24 and 48 hours in the final EC50 -test.

      Response at 24 h     Response at 48 h    
 Concentration Test substance* (mg/l)  Vessel Number  Number Daphnia exposed  Number  % Number 
 Blank-control 10   0  0
 Blank-control  B

 10 

 0  0  0
 0.22  A  10    0  0  0
 0.22  B   10   0  0  0
 0.46  A   10   0  0  0
 0.46  B   10   0  0  0  0
 1.0   10   0  0  0  0
 1.0  B   10   0  0  0
 2.2   10   0  0  20
 2.2   10   0  0  30
 4.6   10   0  0 90 
 4.6  B   10   0  0  9 90 
 10  A   10   0  0  10 100 
 10   10   0  0 10  100 

*Degradation products of test item

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Considering all range-finding and extra tests, it was concluded that during the tests all test substance was converted into hydrolysis products.Quantification of the hydrolysis products was not possible.
The NOEC of the degradation products originating from the test item after 48 hours of exposure was determined to be 1.0 mg/L original product.
The 24h-EC50 of the degradation products of the test item was determined to be >10 mg/L original product.
The 48h-EC50 based on the degradation products corresponded with a nominal test item concentration was calculated to be 2.9 mg/l with a 95% confidence interval between 2.5 and 3.4 mg/l.