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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
other information
Study period:
The study was conducted between 9 August 2011 and 26 August 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing and following a
1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
Validation of mixing period
Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances in the WAF.
A WAF of nominal loading rate of 100 mg/l was prepared, in duplicate, in deionised reverses osmosis water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis (see
Appendix 4).(see in any other infomation and methods section).
Range-finding test
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the range-finding test the test item was prepared as a Water Accommodated Fraction (WAF).
The loading rate to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/l.
Amounts of test item (100 and 1000 mg) were each separately weighed onto a glass slide and suspended in the water column of 10 litres of reconstituted water to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item on the glass slide, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate
75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no globules or micro-dispersions of test item to be present.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 22ºC to 23 ºC with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. The temperatures were measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
Definitive test
Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/l to confirm that no immobilisation or adverse reactions to exposure were observed.

Experimental preparation
Due to the low aqueous solubility and complex nature of the test item for the purposes of the definitive test the test item was prepared as a Water Accommodated Fraction (WAF).
An amount of test item (1000 mg) was weighed onto a glass slide and suspended in the water column of 10 litres of reconstituted water to give the 100 mg/l loading rate. After the addition of the test item, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75 – 100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-particles of test item to be present.
Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 48 hours (see Appendix 5).
Vehicle:
no
Details on test solutions:
Physico-chemical measurements
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a handheld flexi HQ30d meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period.
Total organic carbon analysis
Analysis of the WAF was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control (replicates R1 – R4 pooled) and the 100 mg/l loading rate WAF test group (replicates R1 - R2 and R3 - R4 pooled) at 0 and 48 hours (see Appendix 5)(see in any other information and methods section). Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Evaluation of data
An estimate of the EL*50 values was given by inspection of the immobilisation data.
Test organisms (species):
Daphnia magna
Details on test organisms:
Test Species
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in 150 ml glass beakers containing Elendt M7 medium (see Appendix 1 see in any other information and methods section). in a temperature controlled room at a temperature of 21°C to 22°C. A single temperature was measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test given that was no effect on the reproduction or survival of the stock cultures. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
A positive control (Harlan Laboratories Ltd., Project Number: 41102080) used potassium dichromate as the reference item. Details of the positive control are given in (Appendix 2.see in any other information and methods section) The positive control was conducted between 7 June 2011 and 9 June 2011.


Test Water:
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.


Reconstituted Water
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l
ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:

Not applicable
Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
Temperature was maintained at 21 deg C to 22 deg C throughout the test.
Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.
(See Appendix 4 for results see in any other information and methods section)
Dissolved oxygen:
The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen meter.
Salinity:
Freshwater used.
Details on test conditions:
Exposure conditions
As in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 22ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL not stated
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL not stated
Details on results:
Validation of Mixing Period
Pre-study investigational work (see Appendix 4) (See in any other information on materials and methods section) indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours. Therefore, for the purpose of testing the test item was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Range-finding Test
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1. (see in any other information on results section).
No immobilisation was observed at 10 and 100 mg/l loading rate WAF.
Based on this information, a single loading rate of four replicates, of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilisation or adverse reactions to exposure were observed.

Definitive Test
Immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.(see in any other information on results section).
There was no immobilisation in 20 daphnids exposed to a 100 mg/l loading rate WAF for a period of 48 hours. Inspection of the immobilisation data gave the following results:
Time (h) EL*50 (mg/l)
24 >100
48 >100
The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.

Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 6. (see in any other information on results section). Temperature was maintained at 22ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The temperature was measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion (see Table 3). (see in any other information on results section).

Observations on test item solubility
Observations on the test media were carried out during the mixing and testing of the WAF.
At the start and end of the mixing period and following the 1-Hour standing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless water column with test item visible adhered to the glass slide suspended in the water column. Microscopic inspection of the WAF showed no micro-particles of test item to be present.
At the start and throughout the duration of the test all control and test preparations were observed to be clear colourless solutions.

Total organic carbon analysis
Total Organic Carbon (TOC) analysis of the test preparations at 0 and 48 hours gave measured concentrations of 3.27 and 1.27 mg C/l respectively in replicates R1 – R2 and replicates R3 – R4 at 0 hours and concentrations of 5.32 and 6.07 mg C/l respectively in replicates R1 – R2 and replicates R3 – R4 at 48 hours.
Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.
Results with reference substance (positive control):
Positive Control
A positive control (Harlan Laboratories Ltd., Project No: 41102080) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilisation data by the maximum-likelihood probit method (Finney 1971 ) at 24 and 48 hours based on the nominal test concentrations gave the following results:

Time (h) EC50 (mg/l) 95% Confidence limits
(mg/l)
24 1.5 1.3 - 1.8
48 0.99 0.85 - 1.1
The No Observed Effect Concentration after 24 and 48 hours was 0.56 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item

Table1              Cumulative Immobilisation Data in the Range-findingTest

Nominal Loading Rate

(mg/l)

Cumulative ImmobilisedDaphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

10

0

0

100

0

0

Table2              Cumulative Immobilisation Data in the Definitive Test

Nominal Loading Rate

(mg/l)

Cumulative ImmobilisedDaphnia
(Initial Population: 5 Per Replicate)

24 Hours

48 Hours

No. Per

Replicate

Total

%

No. Per

Replicate

Total

%

Control

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0

100

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0


R1– R4= Replicates 1 to 4

Table3              Vortex Depth Measurements at the Start and End of the Mixing Period

 

Nominal Loading Rate (mg/l)

Control

100

*

+

*

+

Height of Water Column (cm)

26.1

26.1

26.3

26.3

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present


*= Start of mixing period

+= End of mixing period

Appendix 3      Reconstituted Water

i)         Stock Solutions

a)  CaCl2.2H2O                        11.76 g/l
b)  MgSO4.7H2O                        4.93 g/l
c)  NaHCO3                                2.59 g/l
d)  KCl                                         0.23 g/l

ii)        Preparation

An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Appendix 6      Physico-Chemical Measurements

Nominal
Loading Rate
(mg/l)

0 Hours

24 Hours

48 Hours

pH

mg O2/l

%ASV*

T°C

TºC

pH

mg O2/l

%ASV*

T°C

Control

R1

8.0

8.7

100

22

22

8.0

8.4

97

22

 

R2

8.0

8.6

99

22

22

8.0

8.4

97

22

 

R3

8.0

8.6

99

22

22

8.0

8.3

95

22

 

R4

8.0

8.5

98

22

22

8.0

8.4

97

22

100

 

R1

7.9

8.4

97

22

22

8.0

8.4

97

22

R2

8.0

8.4

97

22

22

8.1

8.3

95

22

R3

8.0

8.3

95

22

22

8.0

8.4

97

22

R4

8.0

8.3

95

22

22

8.1

8.3

95

22

 


*ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R1– R4= Replicates 1 to 4

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.
Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item toDaphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l for 48 hours at a temperature of 22ºC under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

The 48-Hour EL*50for the test item to Daphnia magna based on nominal loading rates was greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations at 0 and 48 hours gave measured concentrations of 3.27 and 1.27 mg C/l respectively in replicates R1– R2and replicates R3– R4at 0 hours and concentrations of 5.32 and 6.07 mg C/l respectively in replicates R1– R2and replicates R3– R4at 48 hours.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

*EL = Effective Loading rate

Conclusion

The acute toxicity of the test item to the freshwater invertebrateDaphnia magnahas been investigated and gave a 48-Hour EL*50value of greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.


*EL = Effective Loading rate

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 2009 - 27 August 2009
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
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L, the only concentration tested
- Sampling method: Duplicate samples of the freshly prepared test medium at the start of the test and from aged media at the end of the test. From the control samples, only one of the duplicate samples was analysed from both sampling times.
- Sample storage conditions before analysis: None; samples were measured on day of sampling
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 300.5 mg of the test item was added directly to 3005 mL of the test media and slightly stirred for about 24 hours at room temperature in the dark to dissolve as much of the test item as possible. After cessation of mixing and a following period of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium. - Differential loading: None
- Controls: Untreated test water
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No remarkable observations, clear test medium
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus; clone 5
- Source: In-house laboratory culture. Daphnids 5.5 to 21 hours old at test start were used in the study.
- Feeding during test: None

ACCLIMATION
- Acclimation period: Not required as the test was performed in the same medium as the culturing
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
2.5 mmol (250 mg/L) as CaCO3
Test temperature:
21 °C (test start and end)
pH:
7.9 (test start and end)
Dissolved oxygen:
Test start: 8.1 - 8.8 mg/L
Test end: 8.8 - 8.9 mg/L
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
Nominal: 100 mg/L
Measured: < LOQ
Details on test conditions:
TEST SYSTEM
- Test vessel: Beakers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass with 100 mL volume containing 80 mL of test medium
- Aeration: None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 media was prepared according to the guideline using deionised water and analytical grade salts
- Total organic carbon: < LOQ in control media at test start and test end
- Particulate matter: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: At test start and end pH and dissolved oxygen concentrations were determined in the test media (test concentration and control). Temperature was recorded at test start (test concentration and control) and test end (control).

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16 hours light : 8 hours dark
- Light intensity: 520 - 870 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility after 24 and 48 hours; the appearance of the test media was also assessed at 24 and 48 hours

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY
- Test concentrations: Range finding study performed, test concentrations not reported. No concentrations greater than the limit of solubility were tested.
- Results used to determine the conditions for the definitive study: Yes; 100 mg/L as a limit test selected
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is used twice per year
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobility
Remarks on result:
other: 95 % confidence interval: not determinable
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobility
Remarks on result:
other: 95 % confidence interval: not determinable
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobility
Duration:
24 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobility
Details on results:
- Behavioural abnormalities: None reported
- Other biological observations: None reported
- Mortality of control: 0
- Other adverse effects control: None reported
- Abnormal responses: None reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported
Results with reference substance (positive control):
In the most recent test (performed January 2010; study number 48399220) the EC50 after 24 hours was 1.590 mg potassium dichromate/L indicating that the sensitivity of the Daphnia used in the study.
Reported statistics and error estimates:
No statistical analysis was performed. The EC50 could not be quantified due to the absence of toxicity of the test item. The NOEC and LOEC were determined directly from the raw data.

Table 1: Summary of Biological Results

 Nominal Concentration  % immobilised Daphnia after
 (mg test item/L)  24 hours  48 hours
 Control  0  0
 100  0  0
 EC50 (mg/L)  > 100  > 100
 95 % confidence values (mg/L)  n.d.  n.d.
 NOEC (mg/L) 100 100
 LOEC (mg/L  > 100  > 100

Values refer to nominal test concentrations

n.d. = not determinable

NOEC and LOEC were determined directly from the raw data

Table 2: Influence of the Test Item in test media

 Nominal Concentration

 No. of Daphnia tested

 No. of immobilised Daphnia after

 % of immobilised Daphnia after

 (mg test item/L)

 24 h

 48 h

 24 h

 28 h

 Control

 20

 0

 0

 0

 0

 100

 20

 0

 0

 0

 0

Table 3: Appearance of the Test Item in test media

 Nominal Concentration

 Appearance of the test item in the test media 

 (mg test item/L)

 Start (0 hours)

 24 hours

 End (48 hours)

 100

 0

 0

 0

0: No remarkable observations, clear test medium

Table 4: Dissolved Oxygen and pH-Values in the Test Media

Nominal Concentration 

 Start (0 hours)

 End (48 hours)
 (mg test item/L)  pH  O2 (mg/L)  pH  O2 (mg/L)
Control   7.9  8.8  7.9  8.9
 100  7.9  8.1  7.9  8.8

Table 5: Water temperature in the Test Media at the Start and End of the Test

Nominal Concentration  Water Temperature (°C)
(mg test item/L)  Start (0 hours)  End (48 hours)
 Control  21  21
 100  21  -

Table 6: Results of the Determination of the Test Item in the Test Samples

Sample Description   Total Carbon  Inorganic Carbon   Total Organic Carbon [TOC]
 (mg/L)  age (h)  Found (mg/L)  DF  Calculated (mg Carbon/L)1  Found (mg/L)  DF  Calculated (mg/Carbon/L)1 (mg/Carbon/L)
 Control  0  10.39  1  10.39  8.71  1  8.71  <LOQ
 Control  48  11.88  1  11.88  9.44  1  9.44  <LOQ
 100  0  10.50  1  10.50  8.76  1  8.76  <LOQ
 100  0  10.40  1  10.40  8.74  1  8.74  <LOQ
 100  48  11.75  1  11.75  9.28  1  9.28  <LOQ
 100  48  11.04  1  11.04  9.41  1  9.41  <LOQ

DF: dilution factor

LOQ: Limit of Quantification 3 mg Carbon/L

1rounded values calculated from the exact raw data

Validity criteria fulfilled:
yes
Remarks:
Immobilisation rate in the controls at 48 hours was 0 % and the dissolved oxygen concentration at the end of the test was 8.8 - 8.9 mg/L in the controls and test item concentration; therefore both validity criteria were fulfilled.
Conclusions:
The toxicity of the test item to Daphnia magna was investigated in a guideline study. The 48-hour EC50, based on nominal concentrations, was determined to be > 100 mg/L, the only concentration tested. The 48-hour NOEC and LOEC were determined to be ≥ 100 mg test item/L and > 100 mg test item/L, respectively.
Executive summary:

The toxicity of the test item to Daphnia magna was investigated in a 48-hour immobilisation limit test according to the OECD 202 and EC C.2. guidelines. The study was conducted as a limit test using a water accommodated fraction due to the test item being a complex UVCB. The solutions were prepared by adding 300.5 mg of the test item to 3005 mL of test water and slightly stirring for 24 hours; the aqueous phase was then drawn off and used in the test. A control, containing test water only, was conducted in parallel. Four replicates, each containing five daphnids, of the control and test item concentration were performed. Analysis of the test item concentrations was performed by calibrated TOC analysis at the test start and test end. Immobility of the daphnids in each test item and control replicate was assessed at 24 and 48 hours.

Based on nominal concentrations the 48-hour EC50 for immobilisation was determined to be >100 mg/L, the only concentration tested. The NOEC and LOEC were determined to be ≥100 and >100 mg/L, respectively.

The study is a GLP compliant guideline experimental study fully acceptable for assessment of this endpoint.  

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
other information
Study period:
The study was conducted between 16 August 2011 and 25 August 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing and following a
1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
Validation of mixing period
Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances in the WAF.
A WAF of nominal loading rate of 100 mg/l was prepared, in duplicate, in deionised reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and the concentration of the test item in the 100 mg/l loading rate WAFs and samples taken for Total Organic Carbon analysis (see Appendix 4).

Range-finding test
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the range-finding test the test item was prepared as a Water Accommodated Fraction (WAF).
The loading rate to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/l.
Amounts of test item (100 and 1000 mg) were each separately weighed onto a glass slide and suspended in the water column of 10 litres of reconstituted water to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro-particles of test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate
75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 22ºC to 23ºC with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. The temperatures were measured to be slightly in excess of the 20 ± 1ºC given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive test
Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/l to confirm that no immobilisation or adverse reactions to exposure were observed.

Experimental preparation
Due to the low aqueous solubility and complex nature of the test item for the purposes of the definitive test the test item was prepared as a Water Accommodated Fraction (WAF).
An amount of test item (1000 mg) was weighed onto a glass slide and suspended in the water column of 10 litres of reconstituted water to give the 100 mg/l loading rate. After the addition of the test item, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro particles of test item to be present.
Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 48 hours (see Appendix 5).
Vehicle:
no
Details on test solutions:
Physico-chemical measurements
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi Handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period.

Total organic carbon analysis
Analysis of the WAF was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control (replicates R1 - R4 pooled) and the 100 mg/l loading rate WAF test group (replicates R1 - R2 and R3 - R4 pooled) at 0 and 48 hours (see Appendix 5). Duplicate samples were taken and stored at approximately -20ºC for further analysis if necessary.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in 150 ml glass beakers containing Elendt M7 medium (see Appendix 1) in a temperature controlled room at a temperature of 21ºC to 22ºC. The temperatures were measured to be slightly in excess of the 20 ± 1ºC given in the study plan. This was considered not to affect the results of the test given that no effect on the reproduction or survival of the stock cultures was observed. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
A positive control (Harlan Laboratories Ltd., Project Number: 41102080) used potassium dichromate as the reference item. Details of the positive control are given in Appendix 2. The positive control was conducted between 07 June 2011 and 09 June 2011.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
Temperature was maintained at 21 deg C to 22 deg C throughout the test.
Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.
See Appendix 6 for results.
Dissolved oxygen:
The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen meter.
See Appendix 6 for results.

Salinity:
Fresh water used.
Nominal and measured concentrations:
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/l.
Details on test conditions:
Exposure conditions
As in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21ºC to 22ºC with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL not stated
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL not stated
Details on results:
Validation of Mixing Period
Pre-study investigational work (see Appendix 4) indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours. Therefore, for the purpose of testing the test item was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Range-finding Test
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.
No immobilisation was observed at 10 and 100 mg/l loading rate WAF.
Based on this information, a single loading rate of four replicates, of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilisation or adverse reactions to exposure were observed.

Definitive Test

Immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.
There was no immobilisation in 20 daphnids exposed to a 100 mg/l loading rate WAF for a period of 48 hours. Inspection of the immobilisation data gave the following results:
Time (h) EL*50 (mg/l)
24 >100
48 >100
The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.

Physico-chemical measurements
The results of the physico-chemical measurements are given in Appendix 6. Temperature was maintained at 21ºC to 22ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Some of the temperatures were measured to be slightly in excess of the 20 ± 1ºC given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control group.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion (see Table 3).

Observations on test item solubility
Observations on the test media were carried out during the mixing and testing of the WAF.
At the start and end of the mixing period and following the 1-Hour standing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless water column with test item visible adhered to the glass slide suspended in the water column. Microscopic inspection of the WAF showed no microparticles of test item to be present.
At the start and throughout the duration of the test all control and test preparations were observed to be clear colourless solutions.

Total organic carbon analysis
Total Organic Carbon (TOC) analysis of the test preparations at 0 and 48 hours (see Appendix 5) showed no significant differences in the amount of carbon present within the 100 mg/l loading rate WAF test vessel when compared to the control vessels. Therefore, given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the results gave no evidence of the presence of test item in the WAF.
Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.
Results with reference substance (positive control):
Positive Control
A positive control (Harlan Laboratories Ltd., Project No: 41102080) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilisation data by the maximum-likelihood probit method (Finney 1971 ) at 24 and 48 hours based on the nominal test concentrations gave the following results:
Time (h)
EC50 (mg/l) 95% Confidence limits
(mg/l)
24 1.5 1.3 - 1.8
48 0.99 0.85 - 1.1
The No Observed Effect Concentration after 24 and 48 hours was 0.56 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item
Reported statistics and error estimates:
Evaluation of data
An estimate of the EL*50 values was given by inspection of the immobilisation data.

Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Loading Rate

(mg/l)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

10

0

0

100

0

0

 

Table2              Cumulative Immobilisation Data in the Definitive Test

Nominal Loading Rate

(mg/l)

Cumulative Immobilised Daphnia
(Initial Population: 5 Per Replicate)

24 Hours

48 Hours

No. Per

Replicate

Total

%

No. Per

Replicate

Total

%

Control

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0

100

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0



R1– R4= Replicates 1 to 4

Table3              Vortex Depth Measurements at the Start and End of the Mixing Period

 

Nominal Loading Rate (mg/l)

Control

100

*

+

*

+

Height of Water Column (cm)

26.3

26.3

26.1

26.1

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present


*= Start of mixing period

+= End of mixing period


Appendix 1      Reconstituted Water – Elendt M7 Medium

Solution

Concentration of
stock solution (mg/l)

       (I)           H3BO3

57190

     (II)           MnCl2.4H2O

7210

   (III)           LiCl

6120

  (IV)           RbCl

1420

    (V)           SrCl2.6H2O

3040

  (VI)           NaBr

320

 (VII)           Na2MoO4.2H2O

1260

(VIII)           CuCl2.2H2O

335

  (IX)           ZnCl2

260

    (X)           CoCl2.2H2O

200

  (XI)           Kl

65

 (XII)           Na2SeO3

43.8

(XIII)           NH4VO3

11.5

(XIV)           Na2EDTA.2H2O

5000

     FeSO4.7H2O

1991

An aliquot (dependant on the volume of medium required) of each stock solution was added to a final volume of deionised reverse osmosis water to give stock solution A and stored at approximately 21ºC.

Appendix 1 (continued)   Reconstituted Water – Elendt M7 Medium

Macro Nutrient Stock Solutions

Solution

Concentration of
stock solution (g/l)

           (I)       CaCl2.2H2O

293.80

          (II)      NaHCO3

64.80

        (III)      MgSO4.7H2O

246.60

       (IV)      Na2SiO3.9H2O

50.00

         (V)      KCl

58.00

       (VI)      NaNO3

2.74

     (VII)      K2HPO4

1.84

   (VIII)      KH2PO4

1.43

Vitamin Nutrients

Solution

Concentration of
stock solution (mg/l)

       (IX)      Thiamine hydrochloride

750

      Cyanocobalamine (vitamin

      B12)

10

      D(+) biotin (vitamin H)

7.5

The final medium was prepared by adding an aliquot of stock solution A along with aliquots of each individual Macro Nutrient Stock Solution and an aliquot of the vitamin nutrient to the required amount (final volume) of deionised reverse osmosis water. 

The pH of the prepared media was 8.0 ± 0.2 and stored at approximately 21ºC.

Appendix 3      Reconstituted Water

i)         Stock Solutions

a)  CaCl2.2H2O                        11.76 g/l
b)  MgSO4.7H2O                        4.93 g/l
c)  NaHCO3                                2.59 g/l
d)  KCl                                         0.23 g/l

ii)        Preparation

An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Appendix 4      Validation of Mixing Period

Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic items, in the WAF. A WAF of a nominal loading rate of 100 mg/l was prepared in duplicate in deionised reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis. Samples were taken at 24 hours and 96 hours, each following a 1‑Hour standing period and Total Organic Carbon (DOC) analysis performed. 

The results are summarised as follows:

Test Item Loading Rate

(mg/l)

Time (Hours)

24

96

mg C/l

mg C/l Corrected for Control

mg C/l

mg C/l Corrected for Control

Control

<LOQ

-

<LOQ

-

100

1.82

1.82

2.24

2.24

It is evident from this work that increasing the stirring period did not significantly increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.


 LOQ = Limit of quantitation which was considered to be 1.0 mg C/l

Appendix 5      Total Organic Carbon Analysis

1. METHOD OF ANALYSIS

1.1      Summary

The concentration of Total Organic Carbon (TOC) in the 100 mg/l loading rate WAF was determined by Total Organic Carbon analysis.

1.2      Samples

Samples of the control and the 100 mg/l loading rate WAF test group were taken for analysis at 0 (fresh media) and 48 hours (old media).

1.3      Standards

Standard solutions of potassium hydrogen phthalate (C8H5KO4) were prepared in reverse osmosis water at concentrations of 10, 20, 50 and 100 mg Total Carbon/l. Standard solutions of sodium carbonate (Na2CO3) were prepared in reverse osmosis water at concentrations of 5, 10, 20 and 33.33 mg Inorganic Carbon/l. These solutions were used for calibration of the carbon analyser.

1.4      Procedure

The standard and sample solutions were analysed for TOC using a Shimadzu TOC-VCPHhigh temperature Total Organic Carbon Analyser using the following conditions:

Total Carbon Channel
Temperature                        :          680ºC
Carrier gas                           :          zero grade air
Carrier gas flow rate           :          150 cc/min regulated at 50 psi
Catalyst                                 :          platinum
Injection volume                 :          50 µl


Appendix 5 (continued)   Total Organic Carbon Analysis

Inorganic Carbon Channel
Temperature                        :          ambient
Carrier gas                           :          zero grade air
Carrier gas flow rate           :          150 cc/min regulated at 50 psi
Catalyst                                 :          25% orthophosphoric acid
Injection volume                 :          50 µl

2.        RESULTS

Samples

Nominal Loading Rate

(mg/l)

Concentration of TOC

(mg C/l)

Concentration of TOC Corrected for Control

(mg C/l)

0 Hours

(Fresh Media)

Control R1- R4

<LOQ

-

100 R1- R2

<LOQ

<LOQ

100 R3- R4

<LOQ

<LOQ

48 Hours

(Old Media)

Control R1- R4

<LOQ

-

100 R1- R2

<LOQ

<LOQ

100 R3- R4

<LOQ

<LOQ

R1

 


R1– R4= Replicates 1 and 4

 LOQ = Limit of quantitation which was considered to be 1.0 mg C/l

Appendix 6      Physico-Chemical Measurements

Nominal
Loading Rate
(mg/l)

0 Hours

24 Hours

48 Hours

pH

mg O2/l

%ASV*

T°C

TºC

pH

mg O2/l

%ASV*

T°C

Control

R1

7.9

8.9

100

21

21

7.9

8.8

101

22

 

R2

7.9

8.8

101

22

21

8.0

8.7

100

22

 

R3

7.9

8.8

99

21

21

8.0

8.7

100

22

 

R4

7.9

8.8

101

22

21

8.0

8.7

100

22

100

 

R1

7.9

8.7

100

22

21

7.9

8.7

100

22

R2

7.9

8.6

97

21

21

7.9

8.6

99

22

R3

7.9

8.6

97

21

22

7.9

8.6

99

22

R4

7.9

8.6

97

21

21

8.0

8.6

99

22


*ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R1– R4= Replicates 1 to 4

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.
Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item toDaphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l for 48 hours at a temperature of 21°C to 22°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

The 48-Hour EL*50for the test item toDaphnia magnabased on nominal loading rates was greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations at 0 and 48 hours showed no significant differences in the amount of carbon present within the 100 mg/l loading rate WAF test vessel when compared to the control vessels. Therefore, given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the results gave no evidence of the presence of test item in the WAF.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.


*EL = Effective Loading rate

Conclusion

The acute toxicity of the test item to the freshwater invertebrateDaphnia magnahas been investigated and gave a 48-Hour EL*50value of greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.


*EL = Effective Loading rate

Description of key information

Acute toxicity to Daphnia magna has been assessed for three substances that would fall within this TDI-I category definition. All studies used water accommodated fractions as the test items are poorly soluble UVCBs and all determined EL50 values >100 mg/L, with NOELR values of 100 mg/L. Based on these results, members of the TDI-I category are not expected to be toxic to aquatic invertebrates at the limit of solubility.

Key value for chemical safety assessment

Additional information

Studies were performed to assess the acute toxicity of Diurea 8 and Tetraurea 2 (in base grease) to Daphnia magna (Goodband 2011a,b). In both studies, the method followed was designed to be compatible with the OECD guideline 202, "Daphnia sp, Acute Immobilisation Test". Following preliminary range-finding tests, twenty daphnids (four replicates of five animals) were exposed to a Water Accommodated Fraction (WAF) of Diurea 8 or Tetraurea 2, at a single nominal loading rate of 100 mg/L. The studies were conducted with 48 hours’ exposure at a temperature of 21°C to 22°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

The 48-hour EL50 (effective loading rate) values for both Diurea 8 and Tetraurea 2 were greater than 100 mg/L WAF, based on nominal loading rates. The No Observed Effect Loading Rate (NOELR) values were 100 mg/L WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Total Organic Carbon (TOC) analysis of the test preparations at 0 and 48 hours showed no significant differences in the amount of carbon present within the 100 mg/L loading rate WAF test vessel when compared to the control vessels. Therefore, given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the results gave no evidence of the presence of Diurea 8 or Tetraurea 2 in the WAF. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test substances as a whole, and the dissolved concentrations were below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

In the study Kley and Wydra (2010a), the toxicity of the isolated test item Reaction product of m-tolylidene diisocyanate and cyclohexylamine and cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine to Daphnia magna was investigated in a 48-hour limit test according to the OECD 202 and EC C.2. guidelines. The study was conducted as a limit test using a water accommodated fraction due to the test item being a complex UVCB. Four replicates, each containing five daphnids, were used for the test item and control. Analysis of the test item concentrations was performed by calibrated TOC analysis at test start and test end. The immobility of the daphnids in each test item and control replicate was assessed at 24 and 48 hours.

Based on nominal concentrations the 48-hour EC50 for immobilisation was determined to be >100 mg/L, the only concentration tested. The NOEC was determined to be ≥ 100 mg/L.