3,4-dichlorobenzonitrile

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

december - february 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

Qualifier:

according to guideline

Guideline:

other: NEN-EN-ISO 10712 (1996)

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

- incubation temperature: 23 +/- 1 °C
- incubation time (preliminary suspension): 6 +/- 1 hours
- incubation time (exposure): 16 +/- 2 hours

- Stock solution I: 10.0 g NaNO3, 2.4 g K2HPO4, 1.2 g KH2PO4, 1.0 g yeast extract, dissolved in 500 mL Milli-Q water
- Stock solution II: 10.0 g NaNO3, 2.4 g K2HPO4, 1.2 g KH2PO4, dissolved in 500 mL Milli-Q water
- Stock solution III: 40.0 g D(+)-glucose monohydrate dissolved in 500 mL Milli-Q water
- Stock solution IV: 0.01 g Fe(III)citrate, 4.0 g MgSO4x7H2O, dissolved in 1L Milli-Q water

Test organisms (species):

Pseudomonas putida

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

17 h

Remarks on exposure duration:

in an incubator shaker in the dark

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

17 h

Dose descriptor:

EC10

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Key result

Duration:

17 h

Dose descriptor:

EC50

Effect conc.:

79 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

After incubation extinction values were measured and the cell multiplication inhibition for each test concentration was determined (table 3). Severe toxicity was observed in two of the three vessels at 100 mg/L only. No turbidity was observed in the turbidity controls.

Since it was not clear which of the three vessels at 100 mg/L gave the correct value a second test was performed in which only the three highest concentrations, being 100, 50 and 25 mg/L, were tested. The method and performance of this test were the same as in the first test. The results indicated clear toxicity at 100 mg/L. Therefore, the value I at 100 mg/L in the first test was considered to be an outlying value and excluded from further calculations.

Since no inhibition was seen at 50 mg/L and 87 % inhibition at 100 mg/L, the EC50 -value for growth inhibition was calculated to be approx. 79 mg/L, whereas the EC10 value was approx. 56 mgL.

Table 3: Extinction values of the inoculated dilution series I, II, III and the non-inoculated dilution series t (turbidity control).

concentration 3,4-Dichlorobenzonitrile [mg/L]	extinction in FNU 436 nm						inhibition %
	series			mean	t	corrected mean
	I	II	III	I, II, III
3.1	1.848	1.770	1.656	1.758	0.009	1.749	2.7
6.3	1.770	1.679	1.790	1.746	0.008	1.738	3.3
12.5	1.694	1.771	1.712	1.726	0.007	1.719	4.4
25	1.760	1.776	1.751	1.762	0.007	1.755	2.3
50	1.890	1.927	1.763	1.860	0.007	1.853	-3.2
100	1.592^1)	0.278	0.227	0.253	0.007	0.246	86.9

1) outlying value, excluded from further calculations.

Acceptability of the test:

The EC50 value of 3,5 -Dichlorophenol (reference substance) determined from Pseudomonas putida as calculated by linear regression (Y = 3.1209 X - 18.9803) was 22 mg/L.

The inoculum used in the control (4 FNU) was multiplied by a factor 150 within the test period.

With respect to the accepted range of 10 -30 mg/L for the EC50 of the reference substance and the multiplication factor of the bacteria within the test being over 60, it was concluded that the test conditions were optimal and the results obtained were valid.

Validity criteria fulfilled:

yes

Conclusions:

The EC10 of 3,4-Dichlorobenzonitrile was calculated at 56 mg/l; the EC50 was ca. 79 mg/l.

Executive summary:

3,4 -Dichlorobenzonitrile was investigated for its ability to inhibit the cell multiplication of the bacteria species Pseudomonas putida according to DIN 38412 Part L 8 (March 1991) and NEN-EN-ISO 10712 (1996).

Suspensions of Pseudomonas putida were exposed to concentrations ranging from 3.1 to 100 mg 3,4-Dichlorobenzonitrile per liter. After incubation for 17 h the extinction values of the bacterial cell suspensions were measured. Significant (significant: >10%) toxicity was observed at 100 mg/l only.

Based on the results of the test presently performed, the toxicity threshold value (EC10) of 3,4-Dichlorobenzonitrile was ca. 56 mg/L. The "Bewertungszahl" (Assessment figure or value for bacterial toxicity) is therefore 4.3 (Bewertung wassergefährdender Stoffe, 1989). The EC50 was ca. 79 mg/L.

The EC50 value for the reference substance, 3,5 -Dichlorphenol, tested during the same experiment was 22 mg/L. Since this value was within the accepted range of 10 -30 mg/L for the EC50, and the multiplication factor of the bacteria within the test substance was over 60, it was concluded that the test conditions were optimal and the resluts obtained were valid.

Description of key information

The toxicity threshold value (EC10) and the EC50 value of 3,4 -Dichlorobenzonitrile was determined to be

ca. 56 mg/L and 79 mg/L, respectively.

Key value for chemical safety assessment

EC50 for microorganisms:

79 mg/L

EC10 or NOEC for microorganisms:

56 mg/L

Additional information

3,4 -Dichlorobenzonitrile was investigated for its ability to inhibit the cell multiplication of the bacteria species Pseudomonas putida according to DIN 38412 Part L 8 (March 1991) and NEN-EN-ISO 10712 (1996).

Suspensions of Pseudomonas putida were exposed to concentrations ranging from 3.1 to 100 mg 3,4-Dichlorobenzonitrile per liter. After incubation for 17 h the extinction values of the bacterial cell suspensions were measured. Significant (significant: >10%) toxicity was observed at 100 mg/l only.

Based on the results of the test presently performed, the toxicity threshold value (EC10) of 3,4-Dichlorobenzonitrile was ca. 56 mg/L. The "Bewertungszahl" (Assessment figure or value for bacterial toxicity) is therefore 4.3 (Bewertung wassergefährdender Stoffe, 1989). The EC50 was ca. 79 mg/L.

The EC50 value for the reference substance, 3,5 -Dichlorphenol, tested during the same experiment was 22 mg/L. Since this value was within the accepted range of 10 -30 mg/L for the EC50, and the multiplication factor of the bacteria within the test substance was over 60, it was concluded that the test conditions were optimal and the results obtained were valid.