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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 July 2013 - 13 January 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 or 100 mg/L nominal loading rate
- Sampling method:
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was prepared as a Water Accomodated Fraction. An amount of the test material (2100 mg) was added to the surface of 21 liters of test water to give the 100 mg/L loading rate. After the addition of the test material, the test 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 one hour. A wide bore glass tube, covered at on 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 WAF showed no micro-dispersions or undissolved test material to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL was discarded) to give the 100 mg/L loading rate WAF.
- Eluate: Laboratory tap water dechlorinated and partly softened.
- Differential loading: not applicable
- Controls: Laboratory tap water dechlorinated and partly softened.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Source: Brow Well Fisheries Limited, Hebden, UK
- Age at study initiation: no data - juvenile
- Length at study termination (length definition, mean, range and SD): 4.2 +/- 0.4 cm
- Weight at study termination (mean and range, SD): 0.97 +/- 0.34 g
- Feeding during test: No


ACCLIMATION
- Acclimation period: 24 July to 05 August 2013 (12 days)
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: commercial trout pellets. No data on amount.
- Feeding frequency: No data
- Health during acclimation (any mortality observed): No mortality in the seven days prior to the start of the test
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
140 mg/L as CaCO3
Test temperature:
14-15 °C
pH:
7.9 - 8.1
Dissolved oxygen:
9.6 - 10.0 mg O2/L
Salinity:
Not applicable
Nominal and measured concentrations:
nominal concentrations: 0 or 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 20 L glass exposure vessel
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 20 L glass, no further details
- Aeration: yes, via narrow bore glass tubes.
- Renewal rate of test solution (frequency/flow rate): semi-static system - daily renewal
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): single vessel
- No. of vessels per control (replicates): single vessel
- Biomass loading rate: 0.34 g bw/L

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: 16 h light/ 8 h dark with 20 minute dawn and dusk transition periods
- Light intensity:no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortlaity and sub-lethal effects were recorded at 3, 6, 24, 48, 72 ane 96 h after the start of exposure.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 100 mg/L nminal loading rate
- Results used to determine the conditions for the definitive study:No effects noted in the range finding study
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
There were no mortalities in 7 fish exposed to a 100 mg/L loading rate WAF for a period of 96 hours.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
None

Table 1: Cumulative mortality data in the definitive study.

Nominal loading rate (mg/L)

Cumulative mortality (n = 10)

% mortality

 

3 h

6 h

24 h

48 h

72 h

96 h

96 h

Control

0

0

0

0

0

0

0

100

0

0

0

0

0

0

0

 

The test material concentration in the test samples was initially determined by gas chromatography (GC). Analysis of the control samples from the definitive test showed a peak was present, the profile of which was consistent with that of the test item.

Using this method of analysis, measured concentrations of between 0.0066 and 0.74 mg/L were obtained for the 100 mg/L loading rate WAF.

Given the presence of the peak in the control samples, a second analytical method was developed and the test material concentration in the duplicate test samples was determined by gas chromatography mass spectrometry (GC-MS) using an external standard. The test material gave a chromatographic profile consisting of a single peak and was developed so as to be a more specific method so as to identify the previously seen peaks. The GC-MS profile observed in the control samples was consistent with that of the test material; however it was assessed as a possible trace contaminant which had a similar profile to that of the test material. Given that no adverse effects were observed in the test, it was considered appropriate to correct the test results for the control.

Using this method of analysis, measured concentraions of between less than the concentration seen in the control and 0.60 mg/L were obtained for the 100 mg/L loading rate WAF.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

Validity criteria fulfilled:
yes
Conclusions:
The 96-h LL50 based on nominal loading rates was > 100 mg/L loading rate WAF.
Executive summary:

Introduction

A study was performed under GLP to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, seven fish were exposed to a Water Accommodated Fraction (WAF) of the test material at a single nominal loading rate of 100 mg/L for a period of 96 hours at a temperature of 14 °C under semi-static conditions. the number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results

The test material concentration in the test samples was initially determined by gas chromatography (GC). Analysis of the control samples from the definitive test showed a peak was present, the profile of which was consistent with that of the test item.

Using this method of analysis, measured concentrations of between 0.0066 and 0.74 mg/L were obtained for the 100 mg/L loading rate WAF.

Given the presence of the peak in the control samples, a second analytical method was developed and the test material concentration in the duplicate test samples was determined by gas chromatography mass spectrometry (GC-MS) using an external standard. The test material gave a chromatographic profile consisting of a single peak and was developed so as to be a more specific method so as to identify the previously seen peaks. The GC-MS profile observed in the control samples was consistent with that of the test material; however it was assessed as a possible trace contaminant which had a similar profile to that of the test material. Given that no adverse effects were observed in the test, it was considered appropriate to correct the test results for the control.

Using this method of analysis, measured concentraions of between less than the concentration seen in the control and 0.60 mg/L were obtained for the 100 mg/L loading rate WAF.

Conclusion

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only. Exposure of rainbow trout to the test material gave LL50 values of > 100 mg/Lloading 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 loading rate WAF.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
07 to 11 January 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions were prepared by direct addition of 1000 mg of test material per litre. The test solutions were then stirred thoroughly.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Source: Upwey Trout Farm, Dorset, UK
- Feeding during test: none
- Other: The last medication given to the fish was a 1 mg/l treatment of malachite green on 30 November 1990 (5 weeks prior to the test).

ACCLIMATION
- Acclimation period: 1 week
- Acclimation conditions (same as test or not): same, 15 ± 1°C, glass aquaria
- Type and amount of food: BP Mainstream (batch ref FF123)
- Feeding frequency: The fish was not fed for 24 h prior to the start of the test.
- Health during acclimation (any mortality observed): no mortality
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
37.3 - 38.0 mg/l as CaCO3
Test temperature:
14.4 - 15.2°C
pH:
7.93 - 8.18
Dissolved oxygen:
8.8 - 9.8 mg/l
Nominal and measured concentrations:
Nominal: 0 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 17 L glass vessels with a fill volume of 15 litres
- Aeration: gentle aeration
- Renewal rate of test solution (frequency): every 24 hours
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water supplied from a 100 m³ reservoir with an average retention time of 24 hours. It was
passed through activated carbon, filtered to remove particulate material and dechlorinated with sodium thiosulphate. The treated water was then held in a secondary reservoir with a capacity of 36 m³ and an average retention time of 8 hours. It was delivered to the laboratory via a temperature controlled header tank at a nominal temperature of 15°C.
- Chlorine: < 4 µg/L Cl2
- Alkalinity: 20.4 mg/L as CaCO3
- Conductivity: 138 ± 3 µS/cm
- Intervals of water quality measurement: Daily for pH, dissolved oxygen and temperature.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h dark
- Light: Daylight and artificial lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortalities and symptoms of toxicity at at 24, 48, 72 and 96 hours
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No toxicity in the range of water solubility
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: No toxicity in the range of water solubility
Details on results:
- Other biological observations: In one of the tanks (1000(B)), a partial effect was noted, some of the fish becoming darkly discoloured. This was not significant.
- Mortality of control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test material did not dissolve completely in the dilution water.

No mortalities were observed in the dilution water control and at 1000 mg/L (nominal) during the 96 h exposure. The result was LC50 (96h) is > 1000 mg/L (nominal) and NOEC ≥ 1000 mg/L. Thus, no mortality within the water solubility of the substance was observed under conditions tested.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
04 - 09 April 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accommodated fraction (WAF) were prepared by stirring the test substance in the dilution water for 24 hours to reach equilibrium
concentration. A 20 L complete glass basin was appointed with a glass tube, by what the aqueous phase could be take off witout contamination of the unsolved film of the hydrocarbon. Before the take off of the aqueous phase the preparation was left for 1 hour for complete phase separation.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Source: MPI für Entwicklungsbiologie, Tübingen, Germany
- Length at study initiation (length definition, mean, range and SD): 3.1 - 3.5 cm
- Feeding during test: no

ACCLIMATION
- Acclimation period: At least 12 d
- Health during acclimation (any mortality observed): Mortality ≤ 5%

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Test temperature:
23 ± 2 °C
pH:
8.2 - 8.4
Dissolved oxygen:
> 91%
Nominal and measured concentrations:
Nominal: 10000 mg/L (WAF)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 10 L/ basin
- Aeration: yes, constant with Pasteur pipettes
- Renewal rate of test solution (frequency): every 24 h
- No. of organisms per concentration: 7

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: By aeration (1-2 d) dechlorinated tap water
- Intervals of water quality measurement: Oxygen content, pH-value and temperature were registered after 24 h, 48 h, 72 h and 96 h.

OTHER TEST CONDITIONS
- Photoperiod: 14 light, 10 h dark (Gro-Lux)

EFFECT PARAMETERS MEASURED: Schooling behaviour, activity (mobility), surfacing, floatability
(equilibrium behaviour), dead animals, breathing frequency
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: WAF loading rate
Details on results:
- Behavioural abnormalities: 2 of the 7 test fish showed abnormal behaviour during the 96 h test period. This 2 fish were seperated from the school
and showed lower activity. After 72 h the schooling behaviour of the rest of the fish was also disturbed. The activity and the equilibrium behaviour
was equal to the control fish.
- Other biological observations: The feed ingestion after test end was only reduced at the 2 more reacted fish. All other fish showed the same
behaviour as the control fish.
- Mortality of control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: WAFs showed light turbidity, but
contained no visible emulsion drops.

No mortality of the fish could be observed in the control and the test concentration (10000 mg/l WAF) after 96h under conditions tested. 2 of the 7 test fish showed abnormal behaviour during the 96 h test period. This 2 fish were seperated from the swarm and showed lower activity. But after 72 h the swarm behaviour of the rest of the fish was also disturbed. However, the activity and the equilibrium behaviour was equal to the control fish. The feed ingestion after test end was only reduced at the 2 more reacted fish. All other fish showed the same behaviour as the control fish. As WAFs showed light turbidity and the water solubility of the test substance is < 0.3 mg/l, the abnormal behaviour could cause in unsolved test substance. Overall no mortality could be observed and therefore the LL50 is > 10000 mg/l. Due to the low water solubility (< 0.3 mg/l), no mortality within the water solubility of the test substance were observed and therewith the LC50 (96h) is > water solubility of the test substance.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
no
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The product was added directly in the aquaria after weighting to obtain the desired final concentration.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Oily patches on the surface of the water
Test organisms (species):
Salmo trutta
Details on test organisms:
TEST ORGANISM
- Source: Nordmarka, fish was obtained as fry from a nearby hatchery (OFA) 3 weeks prior to testing.
- Age at study initiation (mean and range, SD): Fry (0+)
- Length at study initiation: 5.5 cm
- Weight at study initiation: 1.64 g
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
11.5 ± 1.0°C
pH:
Start: 6.4 - 6.5
24 hours range (mean): 6.6 - 6.8 (6.8)
Dissolved oxygen:
Start: 100%
24 hours range (mean): 88 - 94% (93%)
Nominal and measured concentrations:
Nominal: 100, 320 and 1000 mg/l
Weighted in (mean): 107, 319 and 1000 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Glass aquarium with 14L water
- Aeration: Water was moderately aerated through a glass diffuser.
- Renewal rate of test solution (frequency/flow rate): Daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.8 g fish/l

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural lake water
- Intervals of water quality measurement: Dissolved oxygen (% saturation) and pH was measured each 24 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The fishes were observed each day and behaviour and condition was
recorded, dead fishes were counted and removed.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Due to expected low toxicity the definitive test was therefore carried out at
only three concentrations.
- Range finding study
- Test concentrations: 100 and 1000 mg/l with 1 fish at each concentration
- Results used to determine the conditions for the definitive study: No mortality was observed after 4 days.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: There was no observable toxic sign related to behaviour or pigmentation.
- Any observations that might cause a difference between measured and nominal values: After 24 h a small amount of "emulsion"
was observed at the two highest concentrations, also some increase in turbidity was noted.

No mortality of the fish could be observed in the control and the test concentrations (100, 320 and 1000 mg/l nominal) during the 96 hours exposure.

After 24 h a small amount of "emulsion" was observed at the two highest concentrations, also some increase in turbidity was noted. Hence, the LC50 (96h) for Salmo trutta exposed to the test substance was > 1000 mg/l (nominal). Due to the low water solubility (< 0.3 mg/l), no effect within the water solubility of the test substance were observed.

Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: analogue substance
Justification for type of information:
Short-term toxicity to fish does not need to be investigated because available data indicate that structural variation does not influence test results or adverse effect profile (see read-across and category justifications attached in Section 13).
Reason / purpose:
read-across source
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality

Description of key information

One reliable study is available. In this study Rainbow trout (Oncorhynchus mykiss) were exposed to a Water Accommodated Fraction of an analogue substance (EC 613 -848 -7) at a nominal loading rate of 100 mg/L under semi-static conditions for 96 hours. The LL50 (96 h) was > 100 mg/L loading rate WAF. The substance is not acutely harmful to fish.

Key value for chemical safety assessment

Additional information

One reliable study is available. In this study (Parr, 2014) performed under GLP according to OECD TG 203 and EC Method C.1, Rainbow trout (Oncorhynchus mykiss) were exposed to the analogue substance under semi-static conditions for 96 hours at a temperature of 14 °C. Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, seven fish were exposed to a WAF of the test material at a single nominal loading rate of 100 mg/L No mortality was observed during the study. The LL50 (96 h) value was > 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

 

The results of this study are supported by data available for the read-across substances Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6)) and its analogues. The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

 

Two semi-static studies, investigating the short-term toxicity to freshwater fish of Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS-No. 11138-60-6), are available. In the first study (Kuttler, 1998) performed according to OECD 203 and using Brachydanio rerio (Danio rerio) the LL50 (96 h) was > 10000 mg/L (WAF loading rate). In the second study (Källqvist and Tobiesen, 1995) with Salmo trutta according to OECD 203 the EC50 (96 h) was > 1000 mg/L and the LOEC > 1000 mg/L. As at the two highest concentrations (320 and 1000 mg/L) a small amount of emulsion and some increase in turbidity was observed, the highest concentration (1000 mg/L) demonstrated a worst case. In both studies no mortality was observed within the water solubility of the test substance under conditions tested. Therefore,it can be concluded that no toxicological effects on fish are expected up to the water solubility for Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS-No. 11138-60-6).

 

There is one study, investigating the short-term toxicity to freshwater fish of Fatty acids, C5-10, esters with pentaerythritol (CAS-No. 68424-31-7). The semi-static study (Combert and Caunter, 1991) with Salmo gairdneri, similar to OECD 203, determined LC50 (96h) > 1000 mg/L (nominal). As the test material did not dissolve completely in the dilution water, the tested concentration demonstrated a worst case. No mortality was observed within the tested concentration (1000 mg/L nominal) during 96h exposure under conditions tested.