Pirimiphos-methyl

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms, other

Remarks:

Pseudomonas growth inhibition test (Bringmann and Kühn; 1980)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 Nov 1988 to 8 Nov 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

other: Bringmann G and Kühn R (1980): Comparison of the toxicity thresholds of water pollutants to bacteria, algae and protozoa in the cell multiplication inhibition test. Water Research 14, 231-241

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The flasks were determined after 6 hours exposure to the test substance.

Vehicle:

no

Details on test solutions:

Deionised water was added to 5 mg of the test substance to give a total volume of 1 L. This was stirred in the dark for 3 days and then allowed to stand in the dark for a further day. Stock solution of 5 mg/L test substance was used for the preparation of the test exposure solution.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

- Test organism: Pseudomonas putida, strain NCIB9494, was obtained as a freeze-dried culture from National Collections of Industrial and Marine Bacteria Ltd, Aberdeen, UK. This was stored at 4°C until use. The freeze-dried culture was rehydrated in 0-5 mL of nutrient broth - A loop of this suspension was streaked onto a nutrient agar slope in a universal bottle - This was incubated at 25°C for 24 hours, and then stored at 4°C , until use as the stock culture

- Preparation of inoculum: 18-20 hours before the start of the test 4 ml of test medium concentrate were added to 46 ml of deionised water in a sterile conical flask. A loop of Pseudomonas putida stock culture was added to this growth medium solution, and then incubated overnight on an orbital shaker (150 rpm). After this period the cells were diluted by addition of fresh growth medium solution at 25 ° C to an optical density was measured. This was used as the test inoculum.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

6 h

Test temperature:

25 °C

pH:

Not reported

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentrations: 1 and 4.5 mg/L of the test substance. See Table 1 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: Flasks
- Type: Closed
- Fill volume: 50 mL
- No. of vessels per concentration (replicates): 2
- No. of vessels per control : 3
- No. of vessels per reference control (replicates): 2
- No. of vessels without inoculum (replicates): 2
- No. of vessels per blank control: 1

TEST MEDIUM
A basal salt solution was prepared by dissolving
- 26.25 g dipotassium hydrogen phosphate,
- 11.25 g potassium dihydrogen phosphate,
- 1.175 g trisodium citrate dihydrate,
- 2.5 g ammonium sulphate and
- 0.25 g magnesium sulphate heptahydrate
in deionised water and making up to 1 litre with deionised water. This solution was autoclaved at 121°C for 15 minutes. A glucose solution was prepared by dissolving 6.25 g glucose in deionised water and making up to 1 L with deionised water solution. This was autoclaved at 121°C for 15 minutes. Equal volumes of the salt solution and the glucose solution were mixed together in a sterile flask to form a growth medium concentrate. This was stored at 4°C until use.

See Table 1 in 'Any other information inmaterial incl. tables' for an overview of the results

Reference substance (positive control):

yes

Remarks:

The reference substance was 3,5 dichlorophenol (99%)

Key result

Duration:

6 h

Dose descriptor:

EC50

Effect conc.:

> 4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Key result

Duration:

6 h

Dose descriptor:

NOEC

Effect conc.:

>= 4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

6 h

Dose descriptor:

EC10

Effect conc.:

> 4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

0% inhibition was found at a concentration of 1 and 4.5 mg/L of the test substance after 6h exposure. It was not possible to test at higher concentrations because of the low water solubility of the substance. See Table 1 in "Any other information in results incl. tables" for an overview of the results.

Results with reference substance (positive control):

94% growth inhibition was found at a concentration of 18 mg/L the reference substance dichlorophenol indicating that the Pseudomonas putida
culture was responding normally to this known toxicant.

Table 1. Optical denisity results

Flask number	Contents (nominal concentration)	Mean optical density at 600nm (4cm cells)	Minus value for uninoculated solution	% Inhibition
1-3	Control	0.553	0.509
4-6	18 mg/L 3,5 dichlorophenol	0.031		94
11,12	4.5 mg/L test substance	0.657	0.626	0
13,14	1 mg/L test substance	0.654	0.624	0
15	Blank uninoculated	0.044
18	4.5 mg/L test substance uninoculated	0.031
19	1 mg/L test substance uninoculated	0.030

Conclusions:

Based on the nominal concentrations, the EC50 and EC10 of the test substance were graphically determined to be >4.5 mg/L. The NOEC was determined to be ≥ 4.5 mg/L

Executive summary:

The toxicity of the test substance was determined by measuring the bacterial growth inhibition of the test substance to the bacterium Pseudomonas putida. The study was conducted as described by Bringmann and Kühn (1980). The bacterium was exposed to nominal concentrations of 1 mg/L and 4.5 mg/L of the test substance for 6 hours. The optical density of each flask content was measured at 600 nm on a spectrophotometer. It was not possible to test at higher concentrations due to the low water solubility of the test substance. The nominal 1 and 4.5 mg/L solutions of the test substance both gave 0% inhibition of growth. Based on these findings, the EC10 and EC50 values were both determined to be >4.5 mg test material per L (nominal). The NOEC was determined to be ≥ 4.5 mg/L

Description of key information

6- h EC50 >4.5 mg/L, Pseudomonas putida, Bringmann and Kühn (1980), Tapp 1989

6- h NOEC ≥4.5 mg/L, Pseudomonas putida, Bringmann and Kühn (1980), Tapp 1989

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

4.5 mg/L

Additional information

The toxicity of the test substance was determined by measuring the bacterial growth inhibition of the test substance to the bacterium Pseudomonas putida. The study was conducted as described by Bringmann and Kühn (1980). The bacterium was exposed to nominal concentrations of 1 mg/L and 4.5 mg/L of the test substance for 6 hours. The optical density of each flask content was measured at 600 nm on a spectrophotometer. It was not possible to test at higher concentrations due to the low water solubility of the test substance. The nominal 1 and 4.5 mg/L solutions of the test substance both gave 0% inhibition of growth. Based on these findings, the EC10 and EC50 values were both determined to be >4.5 mg test material per L (nominal). The NOEC was determined to be ≥ 4.5 mg/L