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Toxicity to soil microorganisms

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Reference
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 4 September 2020
Experimental completion date: 20 October 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
yes
Remarks:
The optimal use-by-date of the organic substrate (Lucerne) was expired at the start of the test that did not disturb the nitrate production.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: GE9061672
- Expiration date of the lot/batch: June 2021
- Purity test date: 100%
- Storage: well-ventilated place, ambient temperature (<30°C) and obscurity

Analytical monitoring:
yes
Remarks:
Analytical Method "CA-MO-006 Analyse des nitrates (NO3-) dans les extraits de sols par IC-UV" (see Appendix 6 attached).
Details on sampling:
Nitrate was extracted from soil by shaking samples with a 1M potassium chloride solution. A ratio of 5 ml of KCl solution per gram dry weight equivalent of soil was used. The mixtures were shaken for at least 1.5 hour at room temperature then let to settle as needed (around 2 hours). The liquid phases were then filtered through 0.8/0.2µm filters and analysed for nitrate.
Vehicle:
yes
Remarks:
Acetone. The solvent was then removed by evaporation before mixing with the soil.
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- A sieved natural soil was amended with powdered plant meal (Lucerne, Medicago sativa) at the recommended lucerne-soil ratio of 5 g of lucerne per kilogram of soil (dry weight); and either treated with the test item or left untreated (blank control).
- Type of organic substrate: soil substrate used for this study was a natural silty sand soil (according to German DIN), provided by Lufa Speyer (Soil type 2.3, batch F2.3 2620).

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The test item is insoluble in water. As a consequence, sand was used as a carrier (as recommended for an optimum distribution of the test substance in soil, a ratio of around 10 g of sand per kilogram of soil dry weight was used): the sand was coated with the test substance dissolved in acetone (22 mL for each treatment). The solvent was then removed by evaporation before mixing with the soil. Equal amounts of sand were added to all treatment levels including the control and the toxic reference.

VEHICLE:
Sand was used as a carrier (as recommended for an optimum distribution of the test substance in soil, a ratio of around 10 g of sand per kilogram of soil dry weight was used): the sand was coated with the test substance dissolved in acetone (22 mL for each treatment). The solvent was then removed by evaporation before mixing with the soil. Equal amounts of sand were added to all treatment levels including the control and the toxic reference.
Total exposure duration:
28 d
Test temperature:
20 ±2 °C
Moisture:
The soil moisture (5.0%) and the Maximal Water Holding Capacity (WHCmax, 31.5%) were determined at the test facility. The soil was not pasteurized nor heat treated; no fertiliser or plant protection products were used. The moisture content of soil samples was maintained during the test: distilled, deionized water was added as needed (at least twice a week, the amount of moisture loss was determined by re-weighing the bottles and adding water to adjust vessels to the starting weight)
Details on test conditions:
TEST SYSTEM
- Testing facility: Groupement de Recherches de Lacq Analytical Department P.O Box 34 64170 LACQ – France
- Test container (type, material, size): Glass beakers of 600 mL of nominal capacity were used for incubation of soils and were covered with perforated polyethylene foil to minimise water loss and to allow gas exchange during the test.
- Amount of soil: 11.2 kg of Lufa 2.3 soil were divided into 8 portions of equal weight of 1.4 kg in which 180 g of water were added. Six of them were mixed with the carrier (sand) containing the test item at the requested test concentration, one was mixed with the carrier without the chemical and the last one was mixed with the carrier and 26.6 g of the toxic reference (to get the required concentration of 20 g/kg sdw). Lucerne (7 g) was then added to each 8 samples and thoroughly mixed.
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: 3


DETAILS OF PREINCUBATION OF SOIL (if any): The test soil was kept refrigerated (around 2 months) prior to use. A pre-incubation was carried out for a period of 48h before the test initiation at a similar test temperature and moisture content that used in the test.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Soil samples were analysed for nitrate at the beginning (day 0) and at the end of the exposure period (28 days). In accordance with the sponsor representative, no intermediate measurement was added.

VEHICLE CONTROL PERFORMED: yes
Nominal and measured concentrations:
As the test item is not an agrochemical, a geometric series of the following nominal concentrations was tested: 31.0, 62.5, 125.0, 250.0, 500.0 and 1000.0 mg/kg sdw (Soil Dry Weight).
Reference substance (positive control):
yes
Remarks:
Sodium Chloride was used to confirm the sensitivity under the test conditions of the soil microorganisms: one single concentration of 20 g/kg sdw was tested with the objective to inhibit the microorganisms activity.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
ca. 31 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: Both parametric and non-parametric statistical tests did not show any significant inhibitory effect up to 1000.0 mg test item/kg soil dw 28 days after application, a NOEC equal to 31.0 mg test item/dw is proposed as a worst case given the raw data obtaine
Results with reference substance (positive control):
- Results with reference substance valid? YES
- Relevant effect levels: Results obtained with the toxic reference confirm the presence of the microorganism activity as it was strongly inhibited at day 28 (the nitrates content was 97.8% lower than the control group).

Reported statistics and error estimates:
Statistical analysis shown failure of data to satisfy the normal distribution and assumption of homogeneity of variances, a non-parametric test was applied and did not determine any significant effect at each treatment level (i.e. NOEC ≥ 1000.0 mg/kg/sdw). However, the 500.0 mg/kg/sdw concentration should be considered as an outlier value due to the high relative standard deviation calculated between all three replicates (>20%). A new statistical analysis was conducted without this treatment: the assumptions of normality and homogeneity of variances were met and the parametric test applied did not determine any significant as well.

Nevertheless, given the observed trend (i.e. nitrates production inhibition at 31.0 and 62.5 mg/kg/sdw treatments then nitrates production stimulation from 125 mg/kg/sdw to the highest tested concentration), the 62.5 mg/kg/sdw concentration should be considered to have significant adverse effects on the bacterial activity as it was observed a decrease of 19.1% of the nitrates production compared to the control. The NOEC is therefore proposed as a worst case to be equal to 31.0 mg test item/kg soil dry weight.

A summary of the results






































Treatment (mg/kg sdw)



% decrease


(means of replicates)



Toxic Reference



-97.8%



31.0



-1.5%



62.5



-19.1%



125.0



3.2%



250.0



27.7%



500.0



27.9%



1000.0



37.4%



Table 3 nitrate-N content determined at Day 0 in control soil












































Replicate



Nitrate-N content (mg/L)



SD



Measure 1



Measure 2



Mean



a



8.82



8.84



8.83



0.017



b



9.71



9.92



9.82



0.150



c



9.20



9.44



9.32



0.167



d



7.03



7.03



7.03



0.006



a, b & c: soil amended with Lucern


d: soil without Lucern


 


Table 4 nitrate-N content determined at Day 28

























































































































































































































































Treatment (mg/kg sdw)



Replicate



Nitrate-N content (mg/L)



SD



RSD


(%)



Deviation of measured contents from mean of controls (%)



Measure 1



Measure 2



Mean



Control



a



56.84



57.04



56.94



0.138



0.2



Per replicate



Mean of replicates



b



53.48



53.54



53.51



0.039



0.1



c



52.20



51.80



52.00



0.277



0.5



Toxic Reference



a



1.80



1.62



1.71



0.129



7.5



-96.8



-97.8



b



0.81



0.79



0.80



0.014



1.7



-98.5



c



0.96



1.03



1.00



0.048



4.8



-98.2



31.0



a



49.24



49.00



49.12



0.175



0.4



-9.3



-1.5



b



55.25



55.30



55.28



0.036



0.1



2.1



c



55.78



55.56



55.67



0.160



0.3



2.8



62.5



a



44.35



43.88



44.12



0.332



0.8



-18.5



-19.1



b



45.24



43.36



44.30



1.330



3.0



-18.2



c



42.82



43.19



43.00



0.264



0.6



-20.6



125.0



a



59.49



59.50



59.50



0.006



0.0



9.9



3.2



b



50.41



49.98



50.20



0.303



0.6



-7.3



c



58.29



57.74



58.01



0.386



0.7



7.1



250.0



a



73.53



74.42



73.97



0.629



0.9



36.6



27.7



b



68.94



69.26



69.10



0.226



0.3



27.6



c



64.15



64.62



64.38



0.330



0.5



18.9



500.0



a



92.93



92.52



92.73



0.287



0.3



71.2



27.9



b



61.81



61.11



61.46



0.494



0.8



13.5



c



53.94



53.34



53.64



0.425



0.8



-0.9



1000.0



a



76.30



74.14



75.22



1.524



2.0



38.9



37.4



b



72.77



73.03



72.90



0.183



0.3



34.6



c



74.82



75.24



75.03



0.296



0.4



38.6



 

Validity criteria fulfilled:
yes
Remarks:
In compliance with guideline OECD 216, the study can be regarded to be valid, since the variation of nitrate-N content between replicate control samples was less than 15% (i.e. 4.7%).
Conclusions:
Since no dose-response relationship could be observed, the EC10, 50 values could not be calculated.

Although both parametric and non-parametric statistical tests did not show any significant inhibitory effect up to the highest tested concentration (i.e. 1000.0 mg test item/kg soil dry weight) 28 days after application, a NOEC equal to 31.0 mg test item/kg soil dry weight is proposed as a worst case given the raw data obtained.
Executive summary:

4,4-isopropylidenediphenol, ethoxylated (BPA-4EO) (EC 500-082-2) was assessed for the purpose of the evaluation of the long-term effects of the test item, after a single exposure, on nitrogen transformation activity of soil microorganisms, following the procedure described in the corresponding OECD 216 guideline.


The test was terminated after 28 days. Soil samples were analysed for nitrate at the beginning (day 0) and at the end of the exposure period (28 days).


Calculation were carried out using the statistical software program ToxRat Professional 3.3.0.


Results obtained with the toxic reference confirm the presence of the microorganism activity as it was strongly inhibited at day 28 (the nitrates content was 97.8% lower than the control group).


However, since no dose-response relationship could be observed, the EC10, 50 values could not be calculated.


Although both parametric and non-parametric statistical tests did not show any significant inhibitory effect up to the highest tested concentration (i.e. 1000.0 mg test item/kg soil dry weight) 28 days after application, a NOEC equal to 31.0 mg test item/kg soil dry weight is proposed as a worst case given the raw data obtained.


In compliance with guideline OECD 216, the study can be regarded to be valid, since the variation of nitrate-N content between replicate control samples was less than 15% (i.e. 4.7%).

Description of key information

4,4-isopropylidenediphenol, ethoxylated (BPA-4EO) (EC 500-082-2) was assessed for the purpose of the evaluation of the long-term effects of the test item, after a single exposure, on nitrogen transformation activity of soil microorganisms, following the procedure described in the corresponding OECD 216 guideline.


The test was terminated after 28 days. Soil samples were analysed for nitrate at the beginning (day 0) and at the end of the exposure period (28 days).


Calculation were carried out using the statistical software program ToxRat Professional 3.3.0.


Results obtained with the toxic reference confirm the presence of the microorganism activity as it was strongly inhibited at day 28 (the nitrates content was 97.8% lower than the control group).


However, since no dose-response relationship could be observed, the EC10, 50 values could not be calculated.


Although both parametric and non-parametric statistical tests did not show any significant inhibitory effect up to the highest tested concentration (i.e. 1000.0 mg test item/kg soil dry weight) 28 days after application, a NOEC equal to 31.0 mg test item/kg soil dry weight is proposed as a worst case given the raw data obtained.


In compliance with guideline OECD 216, the study can be regarded to be valid, since the variation of nitrate-N content between replicate control samples was less than 15% (i.e. 4.7%).

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
31 mg/kg soil dw

Additional information