m-phenylenebis(methylamine)

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The study was conducted between 1995-12-18 and 1995-12-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

no analysis of the test substance

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Analytical monitoring:

no

Details on sampling:

No data

Vehicle:

no

Details on test solutions:

Test water: dechlorinated tap water (by passage through an activated carbon filter)

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Donnington Fish Farm, Upper Swell, Gloucestershire, UK
- Length at study initiation: 4.5 ± 0.2 cm
- Weight at study initiation: 1.23 ± 0.14 g
- Loading: 0.62 g bodyweight/L

ACCLIMATION
- Health during acclimation (any mortality observed): < 1 % in the seven days prior to the study start

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No data

Hardness:

Approximately 100 mg/L as CaCO3

Test temperature:

14 degrees Centigrade

pH:

No data

Dissolved oxygen:

No data

Salinity:

No data

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 20 L
- Aeration: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Direct dispersion into water
- Media renewal: daily

OTHER TEST CONDITIONS
- Medication: None

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Following the initial range-finding study a single test concentration of 100 mg/L in duplicate plus one control were assigned to the definitive study
TEST CONCENTRATIONS
- Range finding study
- Test concentrations: Control, 1.0, 10, 100 mg/L
- Initial population: 3 fish
- Results used to determine the conditions for the definitive study: No mortalities were observed over a period of 96 hours

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

There were no marked reactions to exposure observed in 20 fish exposed to a 100 mg/L test concentration for a period of 96 hours.

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

No data

Sublethal observations / clinical signs:

Cumulative mortality data from a range-finding study conducted between 11 December 1995 and 15 December 1995 are shown in Table 1 below:

Table 1

Concentration mg/L	Cumulative Mortality (initial population = 3)
	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	0
1.0	0	0	0	0
10	0	0	0	0
100	0	0	0	0

Cumulative mortality data for rainbow trout exposed for 96 hours to MXDA are given in the Table 2 (below):

Table 2

Nominal concentration (mg/l)	Cumulative Mortality (initial population : 10)
	3 h	6 h	24 h	48 h	72 h	96 h
Control	0	0	0	0	0	0
100 R1	0	0	0	0	0	0
100 R2	0	0	0	0	0	0

R₁, R₂= replicates 1 and 2

Inspection of the mortality data from the main study provided the results shown in Table 3 based on nominal test concentrations: Table 3
 

Time (hours)	LC50(mg/l)	95% confidence limits (mg/l)
3	>100	-
6	>100	-
24	>100	-
48	>100	-
72	>100	-
96	>100	-

Highest concentration resulting in mortality >= 100 mg/L
Lowest concentration resulting in mortality > 100 mg/L

"No Observed Effect Concentration " (NOEC) >= 100 mg/L

Validity criteria fulfilled:

yes

Conclusions:

There were no marked reactions to exposure observed in 20 fish exposed to a 100 mg/L test concentration for a period of 96 hours.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The definitive study was conducted between 1997-08-11 and 1997-08-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

analysis of test substance is not available to demonstrate that concentrations were satisfactorily maintained

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection 1996-01-22; Date of Signature 1996-02-27

Analytical monitoring:

no

Details on sampling:

No data

Vehicle:

no

Details on test solutions:

For the purpose of the definitive study the test material was prepared by direct dispersion in water. Amounts of test material (0.704, 1.232, 2.20, 3.96 and 7.04 g) were each separately dispersed in dechlorinated tap water and the volume adjusted to 22 litres to give the 32, 56, 100, 180, 320 mg/L test concentrations respectively.

The concentration, homogeneity and stability of the test material in the test solutions were not determined at the request of the sponsor.

The test water used for both the range-finding and definitive studies was the same as that used to maintain the stock fish.

Test organisms (species):

Leuciscus idus

Details on test organisms:

The test was carried out using juvenile golden orfe (Leuciscus idus). Fish were obtained from Xotic Fish Centre, Alfreton, Derbyshire, UK and maintained in-house from 23 June 1997. Fish were maintained in a glass tank with a "single pass" water renewal system. Fish were acclimatised to test conditions from 4 August 1997 to 11 August 1997. The lighting cycle was controlled to give 16 hours light and 8 hours darkness cycle with a 20 minute dawn and dusk transition period.

The stock fish were fed Tetramin® flake food which was discontinued 48 hours prior to the start of the definitive study. There was approximately 3 % mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.8cm (sd= 0.4) and a mean weight 0f 1.97 g (sd=0.62) at the end of the definitive study. Based on the mean weight value this gave a loading rate of 0.90 g bodyweight/litre.

The corpulence factor (K) for the fish used in the study was calculated to comply with the German Water Hazard Classification Scheme from the following equation:

K = [100 * G] / cubic L

where
G = weight of fish
L = length of fish
K = corpulence factor

Hence the corpulence factor (K) = 1.0g/cubic cm

The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 100mg/L as CaCO3.

Test temperature:

The water temperature was controlled at 21 degrees Centrigrade with a dissolved oxygen content of greater than or equal to 8.3 mg O2/L. These parameters were recorded daily.

pH:

Concentration dependent differences in pH were observed throughout the study.

The pH of the control group was observed to vary between 6.9 and 7.7.

Results of the physico-chemical measurements are given in Appendix I (attached).

Dissolved oxygen:

The test vessels received no auxillary aeration. The results of the physico-chemical measurements are given in Appendix I (attached).

Salinity:

No data

Nominal and measured concentrations:

RANGE-FINDING STUDIES

In the initial range-finding study fish were exposed to a series of nominal test concentrations of 1.0, 10 and 100 mg/L.

A second range-finding study was carried out at 32 and 320 mg/L

DEFINITIVE STUDY

Based on the results of the range-finding studies the following test concentrations were assigned to the definitive study: 32, 56, 100, 180 and 320 mg/L.

Details on test conditions:

After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics are shown in Appendix II (attached).

RANGE-FINDING STUDIES

An amount of test material (4.0 g) was dissolved in reverse osmosis water and the volume adjusted to 1 litre to give a 4.0 g/L stock solution. Aliquots (5.5, 55 and 550 mL) of this stock solution were each separately dispersed in dechlorinated tap water and the volume adjusted to 22 litres to give 1.0, 10 and 100 mg/L test concentrations respectively.

After 24 hours exposure sub-lethal effects of exposure were observed at the 100 mg/L test concentration. Therefore a second range-finding study was carried out at 32 and 320 mg/L in an attempt to obtain a No Observed Effect Concentration after 96 hours and 100 % mortalities after 24 hours exposure. Amounts of test material (0.704 and 7.04 g) were each separately dispersed in dechlorinated tap water and the volume adjusted to 22 litres to give 32 and 320 mg/L test concentrations respectively.

For each test concentration 3 fish were added to each test and control vessel which were completely filled with 22 litres of test media to reduce headspace, covered with lids and sealed to reduce evaporation and possible losses of test material due to its suspected volatile nature. The test vessels were maintained at 21 degrees Centigrade in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with a 20 minute dawn and dusk transition period for 96 hours under static test conditions.

The control group was maintained under identical conditions but not exposed to the test material.

After 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.

DEFINITIVE STUDY

As in the range-finding studies, glass exposure vessels were used for each test concentration. At the start of the study 10 fish were placed in each of the test vessels which were completely filled with 22 litres of test media to reduce the headspace, covered with lids and sealed to reduce evaporation and possible losses of test material due to its suspected volatile nature. The test vessels were then maintained at 21 degrees Centigrade in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with a 20 minute dawn and dusk transition period for 96 hours. The test vessels received no auxillary aeration. The fish were not individually identified and received no food during exposure.

The control group was maintained under identical conditions but not exposed to the test material.

A semi-static test regime was employed in the study involving a daily renewal of the test preparations to ensure that the concentrations of the test material remained near nominal and to prevent the build up of nitrogenous waste products.

Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the study. The measurements at zero hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the study after 96 hours, represent those of the used or 24-hour old test preparations.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits = 56-100 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Mortality data
The results of the definitive study showed that the highest test concentration resulting in 0% mortality to be 56 mg/L, the lowest test concentration resulting in 100 % mortality to be 100 mg/L and the No observed effect concentration (NOEC) to be 56mg/L. The NOEC is based upon zero mortalities and the absence of any csub-lethal effects of exposure at this concentration.

The 96-hour LC50 value of 75mg/l corresponds to an evaluation number (Bewertungszahl, BWZ) for the German Water Hazard Classification Scheme of 4.1.

Sub-lethal effects
Sub-lethal effects of exposure were observed at test concentrations of 100 and 180 mg/L. These repsonses were swimming at the surface, loss of equilibrium and the presence of moribund fish.


Physico-chemical measurements
Temperature was maintained at 21 degrees centigrade throughout the study.
There were no treatment related differences for oxygen concentration, concentration dependent differences in pH were observed throughout the study.
The pH of the control group was observed to vary between 6.9 and 7.7. This variation was considered not to affect the validity or integrity of the study given that no mortalities or advserse reactions to exposure were observed in the control group and the Test Guideline states that the pH should not vary by more than 1 unit.

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

The LC50 values and associated confidence limits were calculated by the moving average method of Thompson (1947).

The German Water Hazard Classification Scheme evaluation number (Bewertungszahl, BWZ) is calculated as follows:

The LC50 value is expressed as mg/L x 10E-6 and substituted in the equation below:

Evaluation number = -[log10 (LC50)]

Sublethal observations / clinical signs:

Range Finding Studies

Cumulative mortality data from the exposure of golden orfe to the test material during the range-finding studies are given in Table 1 and sub-lethal effects of exposure are given in Table 2.

Table 1 - Cumulative mortality data in the range-finding studies

Nominal Concentration (mg/L)	Cumulative Mortality (Initial Population = 3)
	24 Hours	48 Hours	72 Hours	96 Hours
Control*	0	0	0	0
1.0	0	0	0	0
10	0	0	0	0
32	0	0	0	0
100	0	2	3	3
320	3	3	3	3

* The results observed for the control groups during both range-finding studies were observed to be identical and therefore were not separately tabulated.

Table 2 - Sub-lethal effects of exposure in the range-finding studies

Nominal Concentration (mg/L)	Sub-lethal Effects*	Time (hours)
		24	48	72	96
Control	No abnormalities detected
1.0	No abnormalities detected
10	No abnormalities detected
32	No abnormalities detected
100	Swimming at the surface	2/3		All Dead
	Moribund	1/3
	Swimming at the bottom		1/1
320	-	All Dead

*Unless stated no adverse reactions were observed

Definitive Study

Cumulative mortality data from the exposure of golden orfe to Meta-xylenediamine during the definitive study are given in Table 3 (below) and the relationship between percentage mortality and concentration is given in Figure 1 (attached).

Table 3 - Cumulative mortality data in the definitive study

Nominal Concentration (mg/L)	Cumulative Mortality (Initial Population = 10)						% Mortality
	3  hours	6    hours	24 hours	48 hours	72 hours	96 hours	96  hours
Control	0	0	0	0	0	0	0
32	0	0	0	0	0	0	0
56	0	0	0	0	0	0	0
100	1	1	6	9	10	10	100
180	2	7	10	10	10	10	100
320	10	10	10	10	10	10	100

Sub-lethal effects of exposure were observed at test concentrations of 100 and 180 mg/L. These responses were swimming at the surface, loss of equilibrium and the presence of moribund fish (see Table 4):

Table 4 - Sub-lethal effects of exposure in the definitive study

Nominal Concentration (mg/L)	Sub-lethal Effects*	Time (hours)
		3	6	24	48	72	96
Control	No abnormalities detected
32	No abnormalities detected
56	No abnormalities detected
100	Swimming at the surface		2/9		1/1	All Dead
	Loss of equilibrium			1/4
	Moribund			1/4
180	Swimming at the surface	1/8		All Dead
	Loss of equilibrium		2/3
	Moribund		1/3
320	-	All Dead

* Unless stated no adverse reactions were observed

Analysis of the mortality data by the moving average method of Thompson (1947) based on the nominal test concentrations gave the following results:

Time (h)	LC50(mg/L)	95% confidence Limits (mg/L)
3	200	170-240
6	150	120-190
24	95	78-120
48	79	71-89
72	75	56-100*
96	75	56-100*

 * Concentrations resulting in 0 and 100% mortalities respectively

The results of the definitive study showed the highest test concentration resulting in 0% mortality to be 56 mg/L, the lowest test concentration resulting in 100% mortality to be 100 mg/L and the No Observed Effect Concentration (NOEC) to be 56 mg/L. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration.

The relationship between the median lethal concentration (LC50) and time is presented graphically in Figure 2 (attached).

The 96 -Hour LC50 value of 75 mg/L corresponds to an evaluation number (Bewertungszahl, BWZ) for the German Water Hazard Classification Scheme of 4.1.

Validity criteria fulfilled:

not specified

Conclusions:

The acute toxicity of the test material to the freshwater fish golden orfe (Leuciscus idus) has been investigated and gave a 96-Hour LC50 value of 75 mg/L with 95% confidence limits of 56-100 mg/L. The No Observed Effect Concentration was 56 mg/L.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

the pH values in the 18.0 mg/L concentration or higher dose groups were 8.6 to 9.4. These values, which are higher than recommended, were attributed to the slightly alkaline nature of the test substance. The study was not judged to have been affected.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Sampling was conducted every 24 hours

Vehicle:

no

Details on test solutions:

Nominal concentrations: Control, 10.0, 18.0, 32.0, 56.0, 100 mg/L

Test organisms (species):

Oryzias latipes

Details on test organisms:

TEST ORGANISM
- Common name: Killifish
- Length at study initiation (length definition, mean, range and SD): 1.88 cm (1.61 - 2.22 cm)
- Weight at study initiation (mean and range, SD): 0.116 g (0.074 - 0.189 g)
- Method of breeding: No fish were wild caught

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

No data

Hardness:

Hardness of dilution water was 60 mg/L

Test temperature:

24 degrees centrigrade ± 1 degrees Centrigrade

pH:

The pH of dilution water was 7.6 at 22 degrees Centigrade

The pH range of the test system was 6.9 to 9.4

Dissolved oxygen:

Dissolved oxygen: 5.1 - 8.4 mg/L

Salinity:

No data

Nominal and measured concentrations:

Concentrations: Control, 10.0, 18.0, 32.0, 56.0, 100 mg/L

Details on test conditions:

Exposure method: semi-static in which the whole quantity of the test solution was exchanged every 24 hours
Test vessel: 5 litre tank with a Teflon sheet covering the surface of the water
Number of fish per vessel: 10
Number of vessels per concentration: 1
Renewal of test solutions: Every 24 hours
Flow rate of test water: 582 L/day
Alkalinity of dilution water: 48mg/L
conductance: 170 uS/cm
Lighting: Indoor light, 16 hours light/8 hours darkness

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

87.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits = 56.0 - >100 mg/L

Details on results:

The substance was stable within the conditions of the test. Measured concentrations were within ± 20% of the nominal concentration for all tes concentrations, therefore, calculation of results was based on the nominal concentrations. Representative chromatograms are attached.

Water quality of dilution water is described in Table A-1 (attached).

Environmental factors which may affect the reliability of the study results: no corresponding events were seen.

The mortality of the control group after 96 hours exposure was zero % and this fulfilled the validity requirement of the study.

Abnormal swimming was observed in the 100 mg/L group. No abnormal symptoms were seen in the control group during the exposure period.

The maximum concentration with 0 % mortality: 56.0 mg/L
The lowest concentration with 100 % mortality: > 100 mg/L

The temperature of the test solution was 24 ± 1 degrees Centigrade for all concentration groups, and the dissolved oxygen was 60% or more of the saturated concentration of the dissolved oxygen (dissolved oxygen concentration at 24.0 degrees Centigrade was 8.25 mg/L). Both of these values fulfilled the study standards.

The pH values for groups exposed to 18.0 mg/L or higher were 8.6 ~ 9.4, which was considered slightly high and attributed to the slight alkaline nature of the test material. The pH values in the other concentration groups and the control group were within the appropriate range (6.0 ~ 8.5).

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

Please refer to Table 3 (below)

Sublethal observations / clinical signs:

The concentrations of the test material in the test solutions were measured at the start of the study and after 24 hours. The results are shown in Table 1:

Table 1 - Measured Concentration of the Test Substance in Test Water

Nominal Concentration mg/L	Measured Concentration mg/L (Percent of Nominal)		Mean* Measured Concentration mg/L
	0 Hr (new)**	24 hour (old)***
Control	<0.02	<0.02	-
10.0	9.99 (100)	10.1 (101)	10.0 (100)
18.00	18.4 (102)	18.3 (102)	18.3 (102)
32.0	32.8 (103)	32.5 (102)	32.6 (102)
56.0	58.2 (104)	58.0 (104)	58.1 (104)
100	103 (103)	103 (103)	103 (103)
*	Geometric mean
**	Freshly prepared test solutions
***	Test solutions after 24 hours exposure

Mortality at each time point is shown in Table 2 (below) and the concentration-mortality curve is shown in Figure 1 (attached):

Table 2 - Mortality of Oryzias Latipes exposed to the test subtance

 

Nominal concentration mg/L	Mean* measured concentration mg/L	Cumulative mortality %
		24 h	48h	72h	96h
Control	-	0	0	0	0
10.0	10.0	0	0	0	0
18.0	18.3	0	0	0	0
32.0	32.6	0	0	0	0
56.0	58.1	0	0	0	0
100	103	0	60	70	70

*geometric mean

 

The median lethal concentration (LC50) was calculated from the data above and the results are shown in Table 3:

Table 3 - Calculated LC50 values

Exposure Period (Hours)	LC50 (mg/L)	95-Percent Confidence Limits (mg/L)	Statistical Method
24	>100	-- ~ --	--
48	92.9	56.0 ~ >100	Binomial
72	87.6	56.0 ~ >100	Binomial
96	87.6	56.0 ~ >100	Binomial

The maximum concentration with zero % mortality and the lowest concentration with 100 % mortality are shown in Table 4:

Table 4: Observation of the highest concentration in zero % mortality and lowest concentration in 100 % mortality

 

Exposure period (hours)	Highest concentration in 0% mortality (mg/L)	Lowest concentration in 100% mortality (mg/L)
24	>100	>100
48	56	>100
72	56	>100
96	56	>100

 

Observed toxic symptoms are shown in Table 5:

Table 5 - Observed Toxicological Symptoms

Nominal Concentration mg/L	Mean* Measured Concentration mg/L	Symptoms (symptom-number of fish)
		24 Hours	48 Hours	72 Hours	96 Hours
Control	---	N	N	N	N
10.0	10.0	N	N	N	N
18.0	18.3	N	N	N	N
32.0	32.6	N	N	N	N
56.0	58.1	N	N	N	N
100	103	AS-10	AS-4	AS-3	AS-3
*	Geometric mean
N	No toxicological symptom was observed
AS	Abnormal swimming

The temperature of the test solution, the concentration of dissolved oxygen and the pH are shown in Tables 6, 7 and 8 respectively:

Table 6 - Temperature

Nominal Concentration mg/L	Temperature °C
	0 Hour	24 Hours		48 Hours		72 Hours		96 Hours
	New*	Old**	new	old	new	old	new	old
Control	23.5	23.6	23.4	23.6	23.5	23.5	23.5	23.6
10.0	23.5	23.6	23.4	23.6	23.5	23.5	23.5	23.6
18.0	23.5	23.6	23.4	23.6	23.5	23.5	23.5	23.6
32.0	23.5	23.6	23.4	23.6	23.5	23.5	23.5	23.6
56.0	23.5	23.6	23.4	23.6	23.5	23.5	23.5	23.6
100	23.5	23.6	23.4	23.6	23.5	23.5	23.5	23.6
*	Freshly prepared solutions
**	Test solutions after 24 hours exposure

Table 7 - Dissolved oxygen concentration

Nominal Concentration mg/L	Dissolved Oxygen Concentration mg/L
	0 Hour	24 Hours		48 Hours		72 Hours		96 Hours
	New*	Old**	new	old	new	old	new	old
Control	8.2	5.4	8.1	6.2	8.4	5.9	8.4	5.8
10.0	8.3	5.2	8.2	6.2	8.4	5.8	8.4	5.1
18.0	8.3	5.2	8.3	6.3	8.4	5.8	8.4	5.2
32.0	8.3	5.1	8.3	6.0	8.4	5.4	8.4	5.3
56.0	8.3	5.5	8.3	6.4	8.4	5.9	8.4	5.8
100	8.2	5.9	8.3	6.8	8.4	7.3	8.4	7.5
*	Freshly prepared solutions
**	Test solutions after 24 hours exposure

Table 8 - pH values

Nominal Concentration mg/L	pH
	0 Hour	24 Hours		48 Hours		72 Hours		96 Hours
	New*	Old**	new	old	new	old	new	old
Control	7.4	7.6	7.5	7.9	7.2	7.4	7.3	6.9
10.0	8.4	7.8	8.3	8.0	8.3	7.9	8.3	7.5
18.0	8.7	8.3	8.6	8.5	8.6	8.5	8.6	8.3
32.0	8.8	8.8	8.9	8.7	8.9	8.8	8.9	8.7
56.0	9.1	9.1	9.2	9.1	9.1	9.1	9.2	9.1
100	9.3	9.3	9.4	9.3	9.4	9.3	9.3	9.4
*	Freshly prepared solutions
**	Test solutions after 24 hours exposure

Conclusions:

The 96 hour LC50 was found to be 87.6 mg/L (95% confidence limits 56.0 - >100 mg/L)

Description of key information

LC50 (96 h) 87.6 mg/L for Oryzias latipes (OECD 203)

LC50 (96 h) 75 mg/L for Leuciscus idus (OECD 203)

LC50 (96 h) > 100 mg/L for Oncorhynchus mykiss (OECD 203)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

87.6 mg/L

Additional information

Two GLP studies are available to describe short-term toxicity to fish. The study conducted by the Japanese Ministry of the Environment (2000) is considered the key study because concentrations of test material were demonstrated to remain within ± 20 % of the nominal. The results reported by Wetton (1998) do not include evidence that test material concentration was satisfactorily maintained because these data were omitted at the request of the sponsor. The third report (Wetton, 1995) is classed as a supporting study due to lack of GLP information and the less precise LC50 value.