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Toxicity to soil microorganisms

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toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 July - 2 September 2005
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Justification for type of information:
Read-across of data measured for the structurally related substances TCPP and TDCP could be considered to be justified. Furthermore, based on the absence of effects in the short-term test in the earthworms, it could be assumed that earthworm would not be the most sensitive species following longer-term exposure, although chronic effects of the substance on the earthworm cannot be ruled out.

according to guideline
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
according to guideline
ISO 14238
GLP compliance:
Analytical monitoring:
Details on sampling:
Nitrate concentration start experiment (Day 0): 28 mg/kg
Details on preparation and application of test substrate:
- Type of organic substrate: sandy loam (Landwirtschaftliche Untersuchungs- und forschungsanstalt Speyer, Germany)

- Method: silica gel containing test substance and 4.3 mL deionized water is added to 50 grams of soil

- Chemical name of vehicle: dichloromethane
- Evaporation of vehicle before use: yes (3 hours in ventilated hood)
Test organisms (inoculum):
other: Nitrifying microorganisms in sandy loam soil
Total exposure duration:
28 d
Test temperature:
18 - 22 degree C
Water added to soil to approximately 45% of the maximum water holding capacity (pF about 2.5)
Details on test conditions:
- Test container: 0.5L serum flasks (glass)
- Amount of soil: 50 g
- No. of replicates: 3

- Standard soil type: sandy loam
- pH (in water): 6.1 - 6.5
- Maximum water holding capacity: 35.2 +/- 3.3 (g/100g)
- Cation exchange capacity (mval/100g): 4 - 14
- Storage: 2 months in refrigerator in polyethylene bags
- Initial microbial biomass as % of total organic C:

Preconditioned by incubating for 7 days at 20 degree C at 10% of its water holding capacity

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Nitrate concentration in soil on day 0 and day 28, activity (mg NO3/kg day), inhibition (%)
Nominal and measured concentrations:
Nominal concentrations test substance: 0, 2, 4, 8, 16, 32, 64 and 128 mg/kg
28 d
Dose descriptor:
Effect conc.:
> 128 mg/kg soil dw
Conc. based on:
other: calculated from measured conc. total phosphorous in stock solution
Basis for effect:
nitrate formation rate
Details on results:
Nitrate formation in soil is not inhibited up to the highest tested concentration of 128 mg/kg soil. The activity of the microorgaisms in soil even increased in the presence of the test substance at the higher concentrations (nitrate concentration in control 53 mg/kg; 54-84 mg/kg in treated soils).
Validity criteria fulfilled:
No inhibition of nitrate formation up to a test concentration of 128 mg/kg soil was observed. Nitrate formation increased where higher concentrations of the test substance were present in the soil (dose-dependent).
Executive summary:

In a 28 -day toxicity study, soil microorganisms were exposed to 2,2 -bis(chloromethyl)trimethylene bis(bis(2 -chloroethyl)phosphate) at 0, 2, 4, 8, 16, 32, 64 and 128 mg/kg soil. There was no inhibition of nitrate formation up to the highest tested concentration of 128 mg/kg soil. Nitrate formation even increased at higher test concentrations. The test substance is considered to be non-toxic to soil microorganisms up to a concentration of 128 mg/kg soil.

28-day NOEC > 128 mg/kg soil


This study is classified as reliable without restrictions and satisfies the guideline requirements for a nitrogen transformation inhibtion test, according to OECD Guideline No.216 (2000).

Description of key information

NOEC of ≥128 mg/kg soil

Key value for chemical safety assessment

Additional information

Inhibition of soil nitrogen transformation by soil micro-organisms has been examined in a study with tris[2-chloro-1-(chloromethyl)ethyl] phosphate (TDCP).  No toxicity data on soil micro-organisms is available for 2,2-Bis(chloromethyl)trimethylene bis[bis(2-chloroethyl)phophate].


Read across from TDCP is considered justified on the grounds that:

- TDCP and 2,2-Bis(chloromethyl)trimethylene bis[bis(2-chloroethyl)phophate] share similar soil adsorption properties (see section 5.4.1). The bioavailability of the substances is therefore expected to be comparable.

- Reliable toxicity data suggest a consistent low order of acute toxicity for 2,2-Bis(chloromethyl)trimethylene bis[bis(2-chloroethyl)phophate] and TDCP to mico-organisms present in waste-water treatment plants (IC50 > 1000 mg/L, IC50 > 10000 mg/L resp, not further reported here)

- Reliable effects data are available for a range of other freshwater and terrestrial organisms for 2,2-Bis(chloromethyl)trimethylene bis[bis(2-chloroethyl)phophate] and TDCP. In no instance does 2,2-Bis(chloromethyl)trimethylene bis[bis(2-chloroethyl)phophate] cause an effects at a lower concentration than TDCP. Therefore it is not anticipated that read-across of this result could result in an overly favourable conclusionin respect of 2,2-Bis(chloromethyl)trimethylene bis[bis(2-chloroethyl)phophate].

Toxicity to micro-organisms

In a 28 -day soil-microorganism nitrogen transformation test according to OECD 216 with TDCP a NOEC of ≥128 mg/kg soil (no inhibition at the highest concentration tested) was determined in the test. 


- European Union Risk Assessment Report "2,2-Bis(chloromethyl)trimethylene bis[bis(2-chloroethyl)phophate]" (V6), CAS 38051-10-4, May 2008

- European Union Risk Assessment Reporttris[2-chloro-1-(chloromethyl)ethyl] phosphate” (TDCP), CAS 13674-87-8, May 2008