Isooctyldinitrophenol

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June 1985 - 28 June 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Work was carried out under supervision according to the procedure herein described and according to the OECD principles of Good Laboratory Practice.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Isooctyldinitrophenol

EC Number:

253-410-9

EC Name:

Isooctyldinitrophenol

Cas Number:

37224-61-6

Molecular formula:

C14H20N2O5

IUPAC Name:

4-(6-methylheptyl)-3,5-dinitrophenol

Details on test material:

- Name of test material (as cited in study report): Dinitro capryl phenol (DNCP)
- Other names: Dinitro octyl phenol
- Batch number: P511.68/85
- Molecular formula: C14H20N2O5
- Molecular weight: 492.03g (inclusive of container - white opaque plastic bottle with a screw cap.)
- Physical state: Brown/Dark Red viscous liquid
- Analytical purity: 89.9%
- Composition of test material, percentage of components: DNCP - 89.9%, MNOP - 4.6%, NDOP - 4.0%, TNP - 1.0%
- Solubility in water: 30mg/l
- pH: 1.8 - 2.0
- Stability - stable, instability caused by overheating
- Intended use - fungicide

Test solutions

Details on test solutions:

Preliminary:
1st test - A concentrated solution containing 30mg/litre of the test substance was prepared in the dilution water.
2nd test - Each exposure concentration was prepared from a concentrated solution containing 5mg/litre of the test substance in the dilution water.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM

The fish used for the preliminary tests were obtained as fry from Lambourn trout, Grove Farm, Newbusy, Berkshire. The fry were reared from eggs imported from the USA on the 19 December 1984 and began to hatch on the 5 January 1985.

The fish used for the definitive test were obtained from Padworth Fisheries, Mill House, Padworth, Berkshire. The fry were reared from eggs imported from Denmark and began to hatch on the 10 April 1985.

The fry were transferred to the WRc Medmneham Laboratory in areated water in a plastic container on 27 March 1985 (preliminary tests) and 11 June 1985 (definitive test). The hardness of the water used to transport the fry, determine at Laboratory on the days of delivery, was 288mg/l as CaCO3 and 262 mg/l as CaCo3 respecitvely.

At the laboratory the fry were held in an aerated flow-through supply of natural groundwater until removed for testing.

The fish were fed with proprietary trout pellets (BP Nutrition Ltd., Mainstream Trout Fry O2) each day during the holding period, at a rate equivalent to 1.5% of the total weight of the fish in the holding tank.

Mortality during the 14-day period prior to transferring fish to the acclimation tank was less than 4% for the preliminary tests.

No mortality occured during the 12-day period prior to transferring fish to the acclimation tank for the definitive test.

ACCLIMATION

The day before the start of the test, fish were removed from the holding tank and placed into acclimation tank, containing aerated water held under static conditions for the preliminary tests and under flow-through conditions for the definitive test. The fish were not fed after transfer to the acclimation tank.

the mean wet weight of individual fish, based ona sample of 10 fish removed at random from the acclimation tank on the 17 and 24 June was 1.75g and 0.93g respectively.

Study design

Test type:

other: Preliminary tests were conducted under static conditions. Definitive tests were carried out under flow-through conditions.

Total exposure duration:

96 h

Test conditions

Hardness:

Water quality measurements taken during the holding period at the laboratory.

27.3.85 - 18.6.85: 254 - 274
11.6.85 - 23.6.85: 264 - 272

Total hardness of the diltution water and of dilutions of the test substance at selected concentrations (low, medium and high exposed concentrations in the definitive test)was measured at the beginning and end of the tests.

Test temperature:

Water quality measurements taken during the holding period at the laboratory.

27.3.85 - 18.6.85: 11.8 - 13.9°C
11.6.85 - 23.6.85: 12.4 - 12.9°C

Tests were carried out in a temperature controlled area at 15 ± 2°C.

Temperature of dissolved oxygen of the dilution water and all dilutions of the test substance was measured before the start of the test and thereafter at 24-hour intervals for the duration of the tests.

pH:

Water quality measurements taken during the holding period at the laboratory.

27.3.85 - 18.6.85: 7.25 - 8.13
11.6.85 - 23.6.85: 7.39 - 7.53

pH of dissolved oxygen of the dilution water and all dilutions of the test substance was measured before the start of the test and thereafter at 24-hour intervals for the duration of the tests.

Dissolved oxygen:

Water quality measurements taken during the holding period at the laboratory.

27.3.85 - 18.6.85: 72 - 101
11.6.85 - 23.6.85: 78 - 94

Concentration of dissolved oxygen of the dilution water and all dilutions of the test substance was measured before the start of the test and thereafter at 24-hour intervals for the duration of the tests.

Nominal and measured concentrations:

Preliminary Tests:

Test 1 (17.6.85 - 18.6.85) : 0.3, 1.0, 3.0, 10 and 30 mg/l.
Test 2 (19.6.85 - 21.6.85) : 0.01, 0.01, 0.1 and 0.3mg/l.

Definitive Test:

0.03, 0.05, 1.0, 1.8 and 3.2 mg/l.

Details on test conditions:

A daily photperiod of 14 hours light, supplied by tungstem filament bulbs, and 10 hours dark with a 30 minute simulation of dawn and dusk was in opration.

DILUTION WATER

The tests were carried ouit using a natural groundwater ontained via a borehole into chalk; the same source as that used to maintain the test organism.

In the preliminary tests, the dilution water was held in 120-litre fibreglass tanks in the test area and gently aerated for a minimum of 12 hours before dilutions of the test substance was prepared.

In the definitive test, the dultion water was taken from the laboratory supply and delivered directly to the dosing apparatus.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

0.07 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL

Effect concentrations 2

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.07 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

0.06 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL

Effect concentrations 4

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.06 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL

Details on results:

Preliminary tests

Mortality data for the test is given in Table 1.

The results indicated that under static conditions the 48-hour LC50 of dinitro capryl phenol (DNCP) to the Rainbow trout was 0.09 mg/l with 95% confidence limits of 0.04 and 0.47mg/l (Table 2).

Within 90 minutes of starting the first test, all of the fish exposed to the test substance at nominal concentrations of 1mg/l and above had died. After 16 hours of exposure, all fish in solutions of the test substance had died.

In the second preliminary test, all of the fish exposed to 0.3mg/l died and three were dead at 0.1mg/l. At the end of the test (48-hours) no further mortalites had occured but one fish at 0.1mg/l was swimming spirally in the test solution.

The dilutions of the test substance used in the first preliminary test were clear, yellow solutions. At the start of the test, the concentration of DNCP in the stock solution was approx. 12.2mg/l, based on absorance determinations. In the second test, the test solutions at 0.1 and 0.3mg/l were tinted slighlty yellow; all other dilutions were clear and colourless and remained so for the duration of the test.

Definitive test

Mortality data for the test is lised in Table 4.

Tables 5 and 6 list the computer calculations of LC50 values at 24, 48, 72 and 96 hours after the start of the test. Concentrations are expressed in milligrams per litre of the test substance as supplied by the sponser.

The LC50 values and 95% confidence limits of the DNCP to the Rainbow trout, calculated using the computer programm of Stephen (2), are give in Text-table 2.

The highest concentration of the test substance causing no mortality and the lowest concentration causing 100% mortality at 96 hours were found to be 0.03mg/l and 0.10mg/l respecitvely.

Dose related effect

Within two hours of starting the test, all of the fish at 0.32mg/l had died and one fish was dead at 0.18mg/l. Approx. 18 hours after the start of the test all of the fish were dead at 0.18mg/l and five fish were dead at 0.1mg/l.

All of the fish exposed to the test substance above 0.05mg/l had died after 24 hours of exposure and one fish at 0.05mg/l was swimming spirally.

48 hours after the start of the test, no further mortalities had occured but two fish were overturned lying immobile on the base of the test vessel at 0.05mg/l.

After 72 hours, two fish had died at 0.05mg/l. At the end of the test (96 hours) no further deaths had occured but one fish at 0.05mg/l was overturned, lying immobile on the base of the vessel.

No adverse effects were exhibited by the fish during the test at 0.03mg/l.

Reported statistics and error estimates:

Wherever appropriate the LC50s (with 95% confidence limits) were calculated by appropriate statistical methods using the number of fish exposed and number dead at each nominal concentration.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Text-table 2: Acute Lethal Toxicity of DNCP to Rainbow Trout.

Period (hours)	LC50 (95% confidence limits) mg/l
24	0.07*
48	0.07*
72	0.06 (0.05 - 0.08)
96	0.06 (0.05 - 0.08)

* estimated value

Table 1: Preliminary test - summary of mortality data

a) First preliminary test

Observation times (hours)	No. dead/exposure concentration mg/l
	Control	0.3	1.0	3.0	10	3 0
1	0	0	1	5	5	5
16	0	5	5	5	5	5

b) Second prelinary test

Observation times (hours)	No. dead/exposure concentrations mg/l
	control	0.01	0.03	0.1	0.3
2	0	0	0	0	1
4	0	0	0	0	3
24	0	0	0	3	5
48	0	0	0	3	5

Number exposed/vessel = 5 fish

Table 2: Second prelimary test: computer calculation of 48-hour LC50.

Control data conc.	Number Exposed	Number Dead	Percent Dead
0.00	5.0	0.0	0.000
Conc.	Number Exposed	Number Dead	Percent Dead	Binomial Prob. (%)
0.30	5.0	5.0	100.000	3.125
0.10	5.0	3.0	60.000	50.000
0.03	5.0	0.0	0.000	3.125
0.01	5.0	0.0	0.000	3.125

The best given information that the binomial can give is that you are 93.75% confident that the LC50 is between 0.03 and 0.30.

Results calculated using moving average method

Span	G	LC50	95% Confidence Limits
3	0.4520571	0.0874	0.0353	0.4685

The probit method requires at least two different concentrations at which the prcent dead is neither 0 nor 100.

Table 4: Definitve test: summary of the mortality data

Observation period (hours)	Nominal exposure concentrations mg/l
	Control	0.03	0.05	0.10	0.18	0.32
2	0 0	0 0	0 0	0 0	0 1	5 5
3	0 0	0 0	0 0	0 0	4 5	5 5
18	0 0	0 0	0 0	3 2	5 5	5 5
21	0 0	0 0	0 0	3 3	5 5	5 5
24	0 0	0 0	0 0	5 5	5 5	5 5
48	0 0	0 0	0 0	5 5	5 5	5 5
72	0 0	0 0	0 2	5 5	5 5	5 5
96	0 0	0 0	0 2	5 5	5 5	5 5

Number exposed / concentration: 10

Number/vessel : 5

Table 5: Definitve test: computer calculation of 24 -hour and 48 -hour LC50 values

Control data conc.	Number Exposed	Number dead	Percent dead
0.00	10.0	0.0	0.000
Conc.	Number Exposed	Number Dead	Percent Dead	Binomial Prob. (%)
0.32	10.0	10.0	100.000	0.098
0.18	10.0	10.0	100.000	0.098
0.10	10.0	10.0	100.000	0.098
0.05	10.0	0.0	0.000	0.098
0.03	10.0	0.0	0.000	0.098

The best information that the binomial test can give is that you are more than 95% confident that the LC50 is between 0.05 and 0.10 thus 0.05 and 0.10 could be used as conservative approximate 95% confidence limits for the LC50.

When the percent dead at all concs. is either 0 or 100, statistically sound LC50 0r 95% confidence limits cannot be calculated by any method. The results of the binomial test can be used to provide conservative approximate 95% confidence limits.

In addition, although not statistically justifed, a useful approximate LC50 can be calulcated as the square root of the product 0f 0.05 and 0.10 which is 0.0707.

Table 6: Definitve test: computer calculation of 72 and 96 hour LC50 values.

Control data conc.	Number Exposed	Number Dead	Percent Dead
0.00	10.0	0.0	0.000
conc.	Number Exposed	Number Dead	Percent Dead	Binomial Prob. (%)
0.32	10.0	10.0	100.000	0.098
0.18	10.0	10.0	100.000	0.098
0.10	10.0	10.0	100.000	0.098
0.05	10.0	2.0	20.000	5.469
0.03	10.0	0.0	0.000	0.098

The best information that the binomial test can give is that you are more than 95% confident that the LC50 is between 0.03 and 0.10 thus 0.03 and 0.10 could be used as conservative approximate 95% confidence limits for the LC50.

Results calculated using moving average method

Span	G	LC50	95% Confidence Limits
3	0.0874320	0.062	0.0496	0.0761

The probit method requires at least 2 different concentrations at which the percent dead is neither 0 nor 100.

Applicant's summary and conclusion

Conclusions:

Discussion and Conclusions

1. The results indicate the under the conditions of the test the 96 -hour LC50 of DNCP to the Rainbow trout was 0.06mg/l, based on the nominal exposure concentration of the substance, with 95% confidence limits of 0.05 and 0.08mg/l.

2. The highest nominal exposure concentration causing no mortality and the lowest nominal concentration causing 100% morality were 0.03mg/l and 0.1mg/l respectively.

3. Dilutions of the test substance at 0.18mg/l and 0.32mg/l were clear but tinted slightly yellow. Below 0.18mg/l, solutions were colourless.