Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-1-2012 2-2-2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study to appropriate guidelines with sufficient justification of deviations due to test substance characteristics. Sufficient substance ID information is available. Restricted use for risk assessment as closed system not entirely relevant to environmental situation.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
see for details Principles of method if other than gudieline.
Principles of method if other than guideline:
The half life of the test chemical was such that degradation products were tested as these were considered most relevant for determining
aquatic hazard. A sealed system was used to ensure maximum possible exposure. A WAF (Water Accommodated Fraction) method was used as the
test substance itself is intentionally mixed with iso dodecane for stability / safety reasons and the active component degrades to multiple breakdown
products all of which could potentially contribute to toxicity. Separation and testing of individual components is therefore not possible.
Quantification of all breakdown products was not possible in the scope of the daphnia test. For this reason the degradation of the active ingredient
was confirmed analytically and the resulting degradation product mixture was tested.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
10 mL of all test concentrations were sampled for analysis at the start of the stirring period (after 15 minutes of rapid stirring) and were pipetted into sample pots on ice that were subsequently transferred to HPLC vials and analyzed for the active peroxide component. Sampling at the end of the
study was not considered required for all concentrations as the rapid disappearance of the test substance has already been demonstrated in
accompanying hydrolysis and algae studies. The highest concentration only was analysed at the end of the stirring period to demonstrate
degredation to the previously investigated degradation products.
Vehicle:
no
Details on test solutions:
The nature of the test chemical being instable and mixed with isododecane require the substance to be tested in a manner that allows all of its
ingredients or resulting degradation products the possibility to dissolve up to their solubility limit in the test media and thus assessing
ecotoxicological effect of all of the components of the test chemical. For this reason a WAF approach was used for this test.
A traditional stock solution and subsequent dilutions were therefore not made during this test.

A WAF was prepared for each test concentration by adding of an accurate amount of the test substance to the test media and allowing to
equilibrate under slow agitation over 24 hours. Each WAF was then considered loaded with the test substance components and / or breakdown
products at their corresponding water solubility limits and was subsequently transferred to test vessels avoiding transfer of any undissolved
material in the WAF. A GLP Hydrolysis and analysis in the GLP algae test for this substance test indicates that the main active ingredient of the test substance was no longer present after 24 hours. Test vessels were completely filled with the resulting WAF solution (essentially a mixture of degradation products, impurities, iso-dodecane and possibly trace levels of peroxide organisms were added and vessels were then sealed for the test duration to maximize exposure and prevent escape of any volatiles formed.

Consideration should be given to the environmental relavance of a closed system for risk assesment purposes. Due to known volatiles being
formed. This approach was used to ensure extreme worst case.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna stock, cultured in conformity with Stan¬dard Operation Procedure (E 1). The animals used in the
test were less than 24 hours old were obtained from parent animals aged between 2-4 weeks. Daphnia were originally obtained from NOTOX B.V.
Hambakenwetering 7 5231 DD ‘s-Hertogenbosch P.O. Box 3476 5203 DL ‘s-Hertogenbosch the Netherlands.

The sensitivity of the Daphnia was checked by performing an inhibition test with a reference compound (potassium dichromate) twice a year. The
sensitivity was tested for compliance with the guidelines.
Test type:
other: Static Sealed
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
ºdH of 11.9
Test temperature:
19.7 to 21.05 °C
pH:
The pH measurements show that the pH remained stable in the test in all concentrations. See results in other information.
Dissolved oxygen:
The oxygen concentration did not fall below 3 mg/l at any point during the test. See results in other information.
Salinity:
-
Nominal and measured concentrations:
Due to degradation products being tested measured initial concentrations are not relevant for expressing the study endpoints. Nominal or loading
concentrations were therefore used :

3.8, 8.4, 18.5, 40.9, and 90 mg/L (closed) were tested
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 ml erlenmeyer with cut glass stop
- Type :closed
.- No. of organisms per vessel:50
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates):4


TEST MEDIUM / WATER PARAMETERS
-The test medium was Dutch Standard Water (DSW), having a pH of approximately 8.2, and a conductivity between 550 and 650 s/cm, containing per liter of de-ionized water: 200 mg of CaCl2•2H2O, 180 mg of MgSO4•7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3 (Standard Operation Procedure O1) . The guideline criteria requires the CaCO3 content to be between 10 and 250 mg/L (approximately 1-14 ºdH). Hardness in the test water was
measured once at the start of the study using the appropriate Dr Lange test kit. This was validated by analyzing a CaCl2 Standard. The dilution was saturated with oxygen before the start of the test. This is done by thorough aeration for at least 30 minutes before use.

OTHER TEST CONDITIONS

- Photoperiod: 16 hours day 8 hours dark
-Highest 2 concentrations required adjustment to that of the natural medium pH of the control


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility

TEST CONCENTRATIONS
- Spacing factor for test concentrations:2.2
- Range finding study: Additional preliminary and supporting studies conducted
- Test concentrations: Initially 100 mg/L in open system resulted in no effect. First definative study 100 mg/L in closed conditions gave 100% effect.
Additionally definative test concentrations (3.8, 8.4, 18.5, 40.9, and 90 mg/L) were repeated with open conditions also resulting in no effect

Previous non GLP research and a GLP hydrolysis study had identified the breakdown products and indicated the short halflife. The test design was
therefore chosen using this information and reccomendations in the "OECD 2000 GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF
DIFFICULT SUBSTANCES AND MIXTURES. (OECD SERIES ON TESTING AND ASSESSMENT NUMBER 23"

Breakdown products include : Isobutyric acid, Isopropanol, propene and acetone
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
3.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Results indicate that in closed conditions the mixture of degradation products of the test chemical in iso dodecane are toxic to daphnia magna.
Due to this result contradicting results found during preliminary studies an additional supporting study was carried out repeating this study using
identical methodology but with open conditions.
Results with reference substance (positive control):
The sensitivity of the Daphnia was checked by performing an inhibition test with a reference compound (potassium dichromate) twice a year. The sensitivity was tested for compliance with the guidelines.
Reported statistics and error estimates:
The Williams test was used for the NOEC/LOEC determination and the Trimmed spearman karber method was used for the EC50 determination.
Normality and variance of the data could not be confirmed. All calculations were conducted using the ToxCalc 5.023 software.

Results Tables

Conc

 

 

 

mg/l

0 hours

24 hours

48 hours

Control I

 5

 5

 5

II

 5

 5

 5

III

 5

 5

 5

IV

 5

 5

 5

Total

 20

 20

 20

3.8    I

 5

 5

2

II

 5

 5

5

III

 5

 5

2

IV

 5

 5

1

Total

20

20

10

8.4    I

 5

 5

0

II

 5

 5

0

III

 5

 5

0

IV

 5

 5

0

Total

20

20

0

18.5    I

 5

5

0

II

 5

5

0

III

 5

4

0

IV

 5

4

0

Total

20

18

0

40.9    I

 5

5

0

II

 5

5

0

III

 5

4

0

IV

 5

5

0

Total

20

19

0

90.0    I

 5

4

0

II

 5

5

0

III

 5

3

0

IV

 5

2

0

Total

20

14

0

pH measurements

Nominal test concentration

(mg/L)

Time (hours)

 

0

48

Control

8.0

7.8

3.8

8.0

7.9

8.4

7.9

7.9

18.5

7.8

7.9

40.9

(7.4)

7.9

7.8

90.0

(7.4)

8.0

7.8

Note : Figures in brackets are values before adjustment with 1 M NaoH

Oxygen Concentration

Nominal test concentration

(mg/L)

Time (hours)

 

0

48

Control

8.8

8.5

3.8

8.9

8.6

8.4

8.9

8.4

18.5

8.9

8.5

40.9

8.9

8.8

90.0

8.9

8.5

Validity criteria fulfilled:
yes
Conclusions:
The study was conducted in a valid manner to GLP and appropriate guidelines. The study may be considered reliable with restrictions due to the
difference in behaviour between open and closed test systems observed. A closed system was used here to ensure maximum exposure and
develop an absolute worst case result. However considering the dramatic difference in results between the two test setups the closed system
results are of debatable relevance for environemntal risk assessment and therefore restricted in there use. Data suggests that volatile degradation
products in combination with the other degradation product illicit an effect in a closed system but not in an open system most likely due to volatiles
leaving the test system. The environmental situation is open and when considering the lack of toxicity in an open system and ready biodegradability
of the formed degradation products it is unlikely that the test chemical will present a risk to aquatic invertibrates.

In conclusion the test chemical has the potential to cause toxicity to invertibrates in closed systems. It is reccomended that the worst case closed
system data is used for C&L and that the risk assesment is based on the supporting data measured in an open system.
Executive summary:

The study was conducted to GLP and to an appropriate guideline with justified modifications and justification for approaches used.validity criteria were met. The data obtained is however contradictory to the preliminary data seen in an open test system. This was further confirmed by an additional supporting study following identical procedures as detailed here but with an open test system.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Conducted as a supporting study. Valid in conjunction with key study only. Not conducted to GLP, No Chemical analysis.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
WAF preparations used and 100 mg /L loading tested in open and sealed conditions
Principles of method if other than guideline:
The test was conducted using OECD 202 as a basis. The test was intended to demonstrate the difference in effect between open and closed
conditions due to the volatile degradation products formed when the parent degrades. A complete concentration range, chemical analysis and full pH and O2 mesurements were not conducted. Additionally this supporting study is intended to clarify the differences observed between preliminary and definitive tests observed in the key study.
GLP compliance:
no
Remarks:
Supporting GLP key study
Analytical monitoring:
no
Details on sampling:
N/A
Vehicle:
no
Details on test solutions:
Identical methods were used to key study. A sealed 100 and 1000 mg/L WAF was prepared and allowed to stir for 24 hours to allow complete
breakdown of the test chemical to its degradation products. The resulting mixture was tested both in closed and open conditions for both
concentrations to allow comparison of effects.


Test organisms (species):
Daphnia magna
Details on test organisms:
Daphnia magna. Juvenile daphnids of D. magna, Clone 5, less than 24 hours old were used in this study obtained from adults between 2 and 4 weeks
old.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
Comparison of Open and Closed Conditions
Test temperature:
20±1 ºC
pH:
See results tables
Dissolved oxygen:
See results tables
Nominal and measured concentrations:
100 mg/L loading and 1000 mg/L loading
Details on test conditions:
4 replicates of 5 animals were used for each test concentration and test system (open or closed).
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Open Conditions
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
< 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Closed System
Details on results:
See results table in any other information for complete illustration significantly higher toxicity was observed in the closed system.
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
N/A
Validity criteria fulfilled:
yes
Remarks:
For supporting purposes Only
Conclusions:
Study should be used in conjunction with the key study only to allow an informed conclusion as to the hazard this substance poses to invertebrates. Clear differences in effect are visible between open and closed systems very likely to due volatile formation which escape in an open system resulting no toxicity. Valid with restrictions as supporting evidence and Invalid as a stand alone study.
Executive summary:

Non GLP supporting study observing the basic OECD202 guidelines modified to investigate the difference in testing in open and closed systems.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Guideline:
other: U.S. EPA Guideline for Testing of Chemicals with Macroinvertebrates and Amphibians EPA-660/3 75-009
Deviations:
not specified
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Daphnia magna
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Nominal and measured concentrations:
Nominal: 0, 15.625, 31.3, 62.5, 125, 250, 500 mg/L
Reference substance (positive control):
not specified
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
51.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% C.I. = 38.28 - 64.22 mg/L

Test conc (mg/L) pH (0 h) pH (48 h) Immobilization* (at 48 hr)
control 8.04 8.06 0 (0)**
15.625 7.83 7.84 0 (0)
31.3 7.61 7.83 0 (0)
62.5 6.8 7.74 16 (80)
125 5.37 5.61 20 (100)
250 4.73 4.89 20 (100)
500 4.28 4.43 20 (100)

*number of daphnids immobilized out of 20

**(#) indicates percentage of daphnids affected in each group

Thus it appears that pH of the unbuffered test solutions is only affected at concentrations above 31 mg/L isobutyric acid. However, since pH controlled and non-pH controlled systems were not simultaneously tested, any effect on toxicity attributable to change in pH cannot be discerned.

Validity criteria fulfilled:
not specified

Description of key information

Due to the extremely short halflife of the test material determined in the hydrolysis study  the degradation products of the test material were deemed themost relevant for aquatic toxicity. For this reason the test material was loaded to the test medium agitated gently for 24 hours and then used directly for testing. All test concentrations were prepared separately as Water Accommodated Fractions to allow the determination of the toxicity of the resulting degradation mixture as a whole to be determined. It is known that the test chemical degrades to multiple degradation products and is itself present in an isododecane solvent. The effects cannot therefore be attributed to a single component with 100% certainty.

However the main degradation product isobutryic acid is the most likely cause of the toxicity observed. A loading of 100 mg/L of test material left to degrade for 24 hours resulted in no visible toxicity to the test organisms in an open system. A 48h-EL50 could therefore not be determined and may be expressed as EL50 >100 mg/L.

In closed conditions the test material was shown to affect invertebrates significantly. In open conditions however no effects were observed

following identical procedures with identical concentrations of test material. A supporting study was conducted testing open and closed conditions in parallel  to allow direct comparison of these conditions. Supporting studies confirmed that the test substance induced no significant effects up to 100 mg/L loading in open conditions but caused significant effects in closed conditions these were however not sufficient to result in classification.  

Public data found for Isobutyric acid are as follows:

Acute Daphnia according to US EPA guideline (EPA-660/3 75 -009, 1975), 48h-EC50 = 51.25 mg/L (SIDS dossier Isobutyric acid, April 2008).

Key value for chemical safety assessment

Additional information

Due to the extremely short half-life of the test material determined in the hydrolysis study the degradation products of the test material were deemed the most relevant for aquatic toxicity. For this reason the test material was loaded to the test medium agitated gently for 24 hours and then used directly for testing. No toxicity to Daphnia magna was observed in open systems. Effects were observed in sealed systems most likely due to volatile breakdown product formation that could not escape. The primary degradation product of the test substance is isobutyric acid and this is itself of low toxicity and is ready biodegradable. The test material should not be considered of concern for this endpoint under environmental conditions as the effects observed in closed conditions are not relevant in the environmental situation.