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Toxicity to soil microorganisms

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Endpoint:
toxicity to soil microorganisms
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From January 26, 2004 to March 09, 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Vehicle:
no
Details on preparation and application of test substrate:
Details on inocculum:
- Nature: soil sample (sandy loam)
- Sampling site:
Data on the history of the site: 2003 fallow soil, 2002 pumpkin, 2001 fallow soil and 400 kg/ha Nitrophoska Spezial, 2000 fallow soil and 400 kg/ha Nitrophoska Spezial, and 1999 cucumber/ leek/ strawberries and 400 kg/ha Nitrophoska Spezial.
- Use pattern: agricultural soil
- Depth of sampling [cm]: 20
- Sand / Silt / Clay content [% dry weight]: Particle sizes according to USDA (%):
< 0.002 mm : 8.5 +/- 1.4;
0.002 – 0.05 mm : 29.2 +/- 3.2
0.05 – 2.0 mm : 62.3 +/- 4.1
- pH: 6.3 +/- 0.4
- Organic carbon content [% dry weight]: 1.02 +/- 0.17
- Nitrogen content [mg/g dry weight]: 0.51
- Cation exchange capacity [mval/100 g]: 10 +/- 2
- Initial microbial biomass: The microbial carbon is 2.3% of the organic carbon present in the soil. More than 1% of the total soil organic carbon
should be biomass-carbon
- Reference of methods: The pH was measured using a pH meter. The temperature was measured and recorded with a thermo couple connected to a data logger.
The amount of inorganic carbon in the absorbers was measured by injecting samples in a Shimadzu TOC apparatus (Shimadzu, s’Hertogenbosch, the Netherlands). Samples were injected in a TOC apparatus. The active microbial biomass was determined according to the respiration method (ISO 1997; Anderson and Domsch, 1978).
- Collection / storage of samples: Sandy loam was purchased from the Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, Speyer,
Germany. The soil was stored in the refrigerator in polyethylene bags until use in the experiment for 10 weeks.
- Preparation of inoculum for exposure: The soil was preconditioned by incubating the soil for 7 d at 20°C at 10% of its water holding capacity
(water content of the soil as received). The soil was amended with powdered alfalfa meal (Medicago sativa), 5 g of alfalfa meal per kilogram of soil (dry weight).
- Pretreatment: No pretreatment
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20°C
Moisture:
The amount of water in the soil was approximately 43% of the maximum water holding capacity. This corresponds with a pF between 2.0 and 2.5.
Details on test conditions:
Details on the test system:
- Culturing apparatus: Glass flasks 0.25 L
- Number of vessels / concentration; 3
- Aeration device: No aeration
- Measuring equipment: Nitrate concentrations were determined with the Reflectoquant, Nitrat Test and RQFlex reflectometer, Merck Darmstadt, Germany.
- Test performed in closed vessels: Yes, to prevent water loss the flasks were covered with an oxygen permeable film.
Nominal and measured concentrations:
0, 50, 100, 200, 400, 800, 1600, 3200 and 6400 mg a.i./kg
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
ca. 70 mg/kg soil ww
Conc. based on:
act. ingr.
Basis for effect:
nitrate formation rate
Remarks on result:
other: calculated
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
ca. 130 mg/kg soil ww
Conc. based on:
act. ingr.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95% confidence limits: 80 and 190 mg a.i./kg;
Details on results:
The activity of the microorganisms transforming nitrogen in soil was slightly inhibited at 50 mg a.i./kg. The EC50 calculated was 130 mg a.i./kg with 95% confidence limits of 80 mg a.i./kg and 190 mg a.i./kg. The EC10, EC20 and EC80 of test substance were 70, 90 and 200 mg a.i./kg respectively. In soil not only formation of nitrate occurs but also reduction of nitrate to nitrogen gas by denitrifying microorganisms. Decrease of the nitrate concentrations in the soil at test substance concentration of 400 mg a.i./kg and higher after 28d is probably the result of the activity of these denitrifying microorganisms. Denitrifying microorganisms are not affected by test substance at concentrations ranging from 400 to 3200 mg a.i./kg whereas the microorganisms responsible of the formation of nitrate are inhibited at these concentrations. The denitrifying microorganisms are inhibited at 6400 mg a.i./kg because after 28d only a limited amount of the nitrate was removed.
Results with reference substance (positive control):
-
Reported statistics and error estimates:
The EC values were computed from the best fitted line (least square method) through the points given by the probit of the percentage inhibition and the logarithm of the concentration of the test substance. The EC10, 25, 50 values were computed from the best fitted line (least-squares method) through the points given by the probit of the percentage inhibition and the logarithm of the concentration of the test substance. Confidence limits were computed on the basis of Fieller's theorem. All computations were performed using the TOXCALC version 5.0 program.

The dose response relationship showed a normal pattern. According to the OECD guidelines the variation between the three control replicates should be less than 15%. This criterion was met because the variation was only 10%. Therefore the test was considered to be valid.

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of the read across study, the 28 d EC50 and EC10 values were determined to be at 130 and 70 mg a.i./kg soil ww (i.e., equivalent to 153 and 83 mg a.i./kg soil dw) respectively.
Executive summary:

A study was conducted to determine the toxicity of the read across substance, C12-16 ADBAC (49.9% active in water) to soil microorganisms, according to OECD Guideline 216, in compliance with GLP. In this study, the inhibition of microbial nitrogen transformation was investigated in sandy loam soil by evaluating the nitrite, nitrate and ammonium formation following 28 d exposure to the read across substance. A volume of 6.04 mL of deionized water containing the read across substance was added to 50-g of soil. The samples were incubated for 7 d at 20°C and at 10% of its water holding capacity. The samples were dosed with read across substance at nominal concentrations 0, 50, 100, 200, 400, 800, 1600, 3200 and 6400 mg a.i./kg soil ww. Analytical dose verification of the stock solutions indicated good correlation with the nominal concentrations. Therefore, doses were presented as nominal concentrations. The nitrogen transformation measurements were carried out at the beginning of the test and at the end at Day 28. The activity of the microorganisms transforming nitrogen in soil was slightly inhibited at 50 mg a.i./kg soil ww. The EC50 calculated was 130 mg a.i./kg siol ww with 95% confidence limits of 80 and 190 mg a.i./kg soil ww. The EC10, EC20 and EC80 of the read across substance were determined at 70, 90 and 200 mg a.i./kg soil ww respectively. In soil not only formation of nitrate occurs but also reduction of nitrate to nitrogen gas by denitrifying microorganisms. Decrease of the nitrate concentrations in the soil was observed at 400 mg a.s./kg soil ww and higher after 28 d. This was probably the result of the activity of these denitrifying microorganisms. The denitrifying microorganisms were inhibited at 6400 mg a.i./kg soil ww, as only a limited amount of the nitrate was removed after 28 d at this concentration. Under the study conditions, the 28 d EC50 and EC10 values were determined to be at 130 and 70 mg a.i./kg soil ww (i.e., equivalent to 153 and 83 mg a.i./kg soil dw) respectively (van Ginkel, 2004). Based on the results of the read across study, similar effect levels can be expected for the test substance.

Endpoint:
toxicity to soil microorganisms
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From July 13, 2000 to October 24, 2000.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5100 (Soil Microbial Community Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Section H.4.1 of the guideline issued by the Netherlands Board for the Authorization of Pesticides (CTB)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not specified
Details on preparation and application of test substrate:
Amendment of soil:
- Type of organic substrate:
(1) Sandy loam - Maasdijk, Heerewaarden, Netherlands
(2) Low humic content sand - Bulb Research Institute, Lisse, Netherlands

Application of the test substance to soil:
- Method: The soil samples were air-dried, sieved to obtain soil granules of less than or equal to 2 mm. Sieved soil samples were analysed for silt, clay, sand, total nitrogen, organic matter and organic carbon content, pH as well as cation exchange capacity by Levington Agriculture, Ipswich, England.

Powdered lucerne meal (C/N ratio 13/1) was added at a concentration of 0.3 g/50 g dw pre-incubated soil. After mixing 50 ± 0.5 g dry weight of soil samples were placed in 100 mL Schott Duran bottles. For each sampling time four replicates at treated and untreated soils were prepared. Samples were treated with test substance at concentrations 0, 10, 100 and 1,000 µg a.i./g soil dw. A stock solution of the test substance was prepared by dissolving 10.0071 g of the test substance in 50 mL of ultra pure water. Further dilutions in ultra pure water were prepared to achieve final test substance concentration series of 10, 100 and 1,000 µg a.i./g soil dw.

The control soils received 0.5 mL of ultra pure water per 50g dw soil. The test substance and control solutions were mixed thoroughly with the soils and the flasks were closed with sponge caps (except during the CO2 measurements) and incubated at 20 ± 2°C for 28d in dark.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 ± 2°C
Moisture:
The moisture content of the soils was adjusted to 40-50% of maximum water holding capacity (MWHC) (i.e., 21.1% for the sandy loam and 15.6% for the low humic content soil).
Details on test conditions:
Test system:
- Testing facility: Levington Agriculture, Ipswich, England.
- Test container (type, material, size): 100 mL Schott Duran bottles
- Amount of soil: 50 ± 0.5 g dry weight of soil
- No. of replicates per concentration: Four
- No. of replicates per control: Four

Soil incubation:
- Method: Bulk / series of individual subsamples: The test substance and control solutions were mixed thoroughly with the soils and the flasks were closed with sponge caps (except during the CO2 measurements) and incubated at 20 ± 2°C for 28 d in dark.

Source and properties of substrate (if soil):
- Geographical reference of sampling site (latitude, longitude):
Sandy loam – Maasdijk, Heerewaarden, Netherlands (51°48'N, 5°22'E)
Low humic content sand – Bulb Research Institute, Lisse, Netherlands (51° 15'N, 4°33'E)

Sandy loam
- % sand: 60
- % silt: 24
- % clay: 16

Low humic content sand
- % sand: 97
- % silt: 1
- % clay: 2

- Soil taxonomic classification: Sandy loam and Sand
- Soil classification system: USDA classification
- pH (in water): Sandy loam: 7.9; Low humic content sand: 6.8
- Maximum water holding capacity (in % dry weigth): MWHC: 21.1% for the sandy loam and 15.6% for the low humic content soil.
- Cation exchange capacity (meq/100 g): Sandy loam: 10.6; Low humic content sand: 4
- Initial microbial biomass as % of total organic C: Sandy loam: 0.9%; Low humic content sand: 0.5%
-Total nitrogen (%): Sandy loam: 0.15%; Low humic content sand: 0.12%

Preincubation of soil (if any): at 20 ± 2°C for 7d

Effect parameters measured (with observation intervals if applicable) : Nitrite, nitrate, ammonium and carbon dioxide formation

Vehicle control: yes, aqueous vehicle control
Nominal and measured concentrations:
Nominal: 0, 10, 100 and 1,000 µg a.i./g soil dw
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 µg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: quantities of nitrate, nitrite and ammonium
Remarks on result:
other: <25% reduction after 28 days (i.e., The difference in CO2 production and nitrogen transformation between the treated and untreated soil samples did not exceed 25% after 28 d of incubation).
Details on results:
The difference in CO2 production and nitrogen transformation between the treated and untreated soil samples did not exceed 25% after 28 d of incubation. The highest inhibition recorded was 82.5% in the nitrite formation rate after 5 d at 10 mg a.i./kg soil dw in the sandy loam soil. After 28 d of incubation, however, no relevant effect was observed (i.e. less than 25% reduction). The test substance was therefore considered to have a low potential for adversely affecting the microbial functions of sandy loam and low humic content sand soils. The highest inhibition recorded during the test was 82.5% inhibition of the nitrification with sandy loam soil after 5 days at 10 µg/kg. However, after 28-days of incubation the inhibition was less than 25%. Therefore, it was not necessary to extend the test beyond 28 day or to perform a final test.

The test substance can be characterised as having no long-term influence on nitrogen and carbon transformations in soils.

Results with reference substance (positive control):
-
Reported statistics and error estimates:
Two tailed Dunnett test (multiple comparison with a control).

For result tables, kindly refer to the attached background material section of the IUCLID.

Validity criteria fulfilled:
yes
Remarks:
The variation between replicate control samples was less than ±15%, which means that the results are within the validity criteria stated in the guidelines.
Conclusions:
Under the conditions of the study, the read across substance was therefore considered to have a low potential for adversely affecting the microbial functions of sandy loam and low humic content sand soils and the 28 d EC50 and NOEC were considered to be at >1000 and ≥1000 µg a.i./g soil dw respectively.
Executive summary:

A study was conducted to determine the toxicity of the read across substance, C12-16 ADBAC (49-51% active in water) to soil microorganisms, according to OECD Guideline 216 and 217, and US EPA OPPTS 850.5100, in compliance with GLP. In this study, the effects of the read across substance on carbon mineralization and nitrogen transformation activity of soil micro-organisms were investigated in two soil types (sandy loam soil and a low humic content sand) by evaluating nitrite, nitrate, ammonium and carbon dioxide formation following 28 d exposure. Fifty grams dry weight of soil samples were mixed with lucerne meal (13:1 carbon:nitrogen) and placed in 100 mL bottles. The samples were incubated in the dark at 20±2°C for 28 d. The moisture content of the samples was checked weekly. The samples were dosed with read across substance at nominal concentrations 0, 10, 100 and 1000 µg a.i./g soil dw. No analytical dose verification was performed for the read across substance. Samples were taken to determine nitrogen metabolite content on days 5 and 28 and the CO2 evolution was determined on Days 5 – 8 and 25 – 28. No significant reduction in ammonium formation was observed. The difference in the CO2 production and nitrogen transformation between the treated and untreated soil samples did not exceed 25% after 28 d of incubation. The highest inhibition recorded was 82.5% in the nitrite formation rate after 5 d at 10 mg a.i./kg soil dw in the sandy loam soil. After 28 d of incubation, however, no relevant effect was observed (<25% reduction). Therefore, it was not necessary to extend the test beyond 28 d. Under the conditions of the study, the read across substance was therefore considered to have a low potential for adversely affecting the microbial functions of sandy loam and low humic content sand soils and the 28 d EC50 and NOEC were considered to be at >1000 and ≥1000 µg a.i./g soil dw respectively (de Vette, 2001). Based on the results of the read across study, similar effect levels can be expected for the test substance.

Description of key information

In line with the C12 -16 biocides assessment report and based on the results of the read across study, the lower 28d EC50 = 153 mg a.i./kg dw and a 28d EC10 = 83 mg a.i./kg dw soildue to inhibition of microorganisms has been considered further for hazard/risk assessment.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:
153 mg/kg soil dw
Long-term EC10 or NOEC for soil microorganisms:
83 mg/kg soil dw

Additional information

Study 1. A study was conducted to determine the toxicity of the read across substance, C12-16 ADBAC (49.9% active in water) to soil microorganisms, according to OECD Guideline 216, in compliance with GLP. In this study, the inhibition of microbial nitrogen transformation was investigated in sandy loam soil by evaluating the nitrite, nitrate and ammonium formation following 28 d exposure to the read across substance. A volume of 6.04 mL of deionized water containing the read across substance was added to 50-g of soil. The samples were incubated for 7 d at 20°C and at 10% of its water holding capacity. The samples were dosed with read across substance at nominal concentrations 0, 50, 100, 200, 400, 800, 1600, 3200 and 6400 mg a.i./kg soil ww. Analytical dose verification of the stock solutions indicated good correlation with the nominal concentrations. Therefore, doses were presented as nominal concentrations. The nitrogen transformation measurements were carried out at the beginning of the test and at the end at Day 28. The activity of the microorganisms transforming nitrogen in soil was slightly inhibited at 50 mg a.i./kg soil ww. The EC50 calculated was 130 mg a.i./kg soil ww with 95% confidence limits of 80 and 190 mg a.i./kg soil ww. The EC10, EC20 and EC80 of the read across substance were determined at 70, 90 and 200 mg a.i./kg soil ww respectively. In soil not only formation of nitrate occurs but also reduction of nitrate to nitrogen gas by denitrifying microorganisms. Decrease of the nitrate concentrations in the soil was observed at 400 mg a.s./kg soil ww and higher after 28 d. This was probably the result of the activity of these denitrifying microorganisms. The denitrifying microorganisms were inhibited at 6400 mg a.i./kg soil ww, as only a limited amount of the nitrate was removed after 28 d at this concentration. Under the study conditions, the 28 d EC50 and EC10 values were determined to be at 130 and 70 mg a.i./kg soil ww (i.e., equivalent to 153 and 83 mg a.i./kg soil dw) respectively (van Ginkel, 2004).

Study 2. A study was conducted to determine the toxicity of the read across substance, C12-16 ADBAC (49-51% active in water) to soil microorganisms, according to OECD Guideline 216 and 217, and US EPA OPPTS 850.5100, in compliance with GLP. In this study, the effects of the read across substance on carbon mineralization and nitrogen transformation activity of soil micro-organisms were investigated in two soil types (sandy loam soil and a low humic content sand) by evaluating nitrite, nitrate, ammonium and carbon dioxide formation following 28 d exposure. Fifty grams dry weight of soil samples were mixed with lucerne meal (13:1 carbon:nitrogen) and placed in 100 mL bottles. The samples were incubated in the dark at 20±2°C for 28 d. The moisture content of the samples was checked weekly. The samples were dosed with read across substance at nominal concentrations 0, 10, 100 and 1000 µg a.i./g soil dw. No analytical dose verification was performed for the read across substance. Samples were taken to determine nitrogen metabolite content on days 5 and 28 and the CO2 evolution was determined on Days 5 – 8 and 25 – 28. No significant reduction in ammonium formation was observed. The difference in the CO2 production and nitrogen transformation between the treated and untreated soil samples did not exceed 25% after 28 d of incubation. The highest inhibition recorded was 82.5% in the nitrite formation rate after 5 d at 10 mg a.i./kg soil dw in the sandy loam soil. After 28 d of incubation, however, no relevant effect was observed (<25% reduction). Therefore, it was not necessary to extend the test beyond 28 d. Under the conditions of the study, the read across substance was therefore considered to have a low potential for adversely affecting the microbial functions of sandy loam and low humic content sand soils and the 28 d EC50 and NOEC were considered to be at >1000 and ≥1000 µg a.i./g soil dw respectively (de Vette, 2001).

Based on the above studies, same effect levels and low toxicity potential were concluded in the biocide assessment report on C12-16 ADBAC by RMS Italy. They further stated that: “The studies from the two dossiers, although all rated 1, show marked difference in the results, even when the soil characteristics were similar like in the case of tests conducted with sandy loam soils. The endpoint with the lowest values is therefore selected to be taken into account, i.e., 28d EC50 = 153 mg a.i./kg dw (130 mg/kg wwt soil) and a 28d EC10 = 83 mg a.i./kg dw soil (70 mg a.i./kg ww soil), retrieved from the EQC dossier.”(ECHA biocides assessment report, 2015). Similar conclusions were drawn in the Coco TMAC biocides assessment report, 2016, where the endpoint was mainly assessed based on read across to DDAC along with the EQC owned supporting study on C12-16 ADBAC. The lowest 28d EC50 = 101.3 mg a.s. /kg dw (corrected for MW) and 28d EC10 = 59.3 mg a.s. /kg dw (corrected for MW) from the study on DDAC was selected for risk assessment.

In line with the C12 -16 biocides assessment report and gven that the read across to C12-16 ADBAC can be justified for the test substance based on a category approach, the lower 28d EC50 = 153 mg a.i./kg dw and a 28d EC10 = 83 mg a.i./kg dw soil due to inhibition of microorganisms has been considered further for hazard/risk assessment.