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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:
From 28 january 2016 (signature of the study plan) to 10 June 2016 (reception of the final report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
12th november 2014
Analytical monitoring:
no
Vehicle:
yes
Remarks:
deionised water
Details on preparation and application of test substrate:
PREPARATION OF TEST TREATMENTS
A water suspension of the Supragil WP was prepared at a nominal concentration of 50 mg/mL by direct addition of Supragil WP (500 mg) to 100 mL of deionised water. This preparation was mixed gently and ultrasonicated for 10 minutes to add suspension. This suspension was serially diluted to prepare solutions to dose the soil at 1000, 500, 100, 10 and 1.0 mg Supragil WP/kg dry soil treatment levels.

Solution
Reference Treatment concentration
(mg Supragil WP./kg dry soil) Preparation Details
A 1000 5000 mg of Supragil WP made up to 100 mL
B 500 50 mL A made up to 100 mL with deionised water
C 100 20 mL B made up to 100 mL with deionised water
D 10 10 mL C made up to 100 mL with deionised water
E 1.0 10 mL D made up to 100 mL with deionised water


AMENDMENT OF SOIL
A series of 1500 g (dry weight equivalent) aliquots of the pre-acclimated soil was weighed out into a stainless steel mixing bowl.
The test soil was amended with dry powdered lucerne at a rate of 2.5 g of lucerne/500 g of soil (dry weight equivalent) to the control and treatment groups by hand mixing prior to treatment with the test solutions.
The lucerne had a carbon content of 45.21% w/w and a nitrogen content of 3.14% w/w giving a C:N ratio of 14.4:1 (determined by Butterworth Laboratories Ltd, Teddington, Middlesex, TW11 8NY, United Kingdom, and reported in a separate GLP compliant Study report ID 1404-0246).

APPLICATION OF TEST SUBSTANCE TO SOIL
The treatment solutions in water (20 mL dose volume/kg dry soil) were then added to the soil and the volume of water to achieve a test moisture of 42 % of the MWHC. The deionised water control soil was treated with the same quantity of deionised water only. The bulk dosed amended soil was mixed in a planetary mixer (Crypto Peerless, Halifax, UK) and then divided equally into replicate test vessels. Treatments were prepared in the following order: deionised water control followed by the test item in ascending rates.

VEHICLE: yes (water)





Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 ± 2 °C
Moisture:
The MWHC of the soil was determined as 27.6 %. Soil moisture levels for the study were maintained at 42 % +/- 5% of the MWHC of the soil.
Details on test conditions:
TEST ITEM
- Testing facility:
- Test container (type, material, size): 2.0 L plastic container (internal dimensions 16.7cm x 16.7cm x 9cm deep) with the lid perforated with a single 3 mm diameter hole to ensure aerobic conditions.
- Amount of soil: 500 g (dry weight equivalent) of soil
- No. of replicates per concentration: 3
- No. of replicates per vehicle control: 3

SOIL INCUBATION
- Method: The bulk dosed amended soil was mixed in a planetary mixer (Crypto Peerless, Halifax, UK) and then divided equally into replicate test vessels. Treatments were prepared in the following order: deionised water control followed by the test item in ascending rates.

SOURCE AND PROPERTIES OF THE SOIL
- Single, common agricultural soil as described in OECD Guideline 216 (2000). The soil used was LUFA standard soil type 2.3 (Batch number F2.34914) and was supplied by LUFA-Speyer, Obere Langgasse 40, 67346 Speyer, Germany.
- The soil sampling was carried out according to ISO 10381-6 (1993) as recommended in OECD 216 (2000).
- Soil was received at CEMAS on the 15 December 2015 and was stored in the dark in a refrigerator (nominally at 4°C) until required.
- The test soil had a sand content of 59.7 %, a pH of 6.8 (measured in water), with an organic carbon content of 0.67 % and a microbial biomass (198 mg Cmic/kg) of not less than 1 % of the total organic carbon content (actual value 2.96 %). In most cases, a soil with these characteristics represents a worst-case situation, since adsorption of the test chemical to soil is minimal and consequently its availability to the microflora is maximal.
- Acclimation of the soil used for the study was started on 17 February 2016, when the soil was brought to a moisture content of 35 % ( 5 %) of the maximum water holding capacity (MWHC) of the soil, and left to acclimate in a test cabinet in the dark at 20 ± 2°C for five days before treatment.

EXPERIMENTAL CONDITIONS
The vessels were maintained in the dark at 20°C +/- 2°C. Temperature was monitored throughout the study.
The products of the process of mineralisation were extracted from the soil on Days 0 and 28 after treatment and incubation at 20°C +/- 2°C.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The extracted solutions were analysed for nitrate, nitrite and ammonium nitrogen concentrations from the samples taken on Day 0 and Day 28 and the results converted to mg/kg dry soil basis.

VEHICLE CONTROL PERFORMED: yes (water)

RANGE-FINDING STUDY
- Test concentrations: Water Control
0.10, 1.00, 10.0, 100 and 1000 mg Supragil WP/kg dry soil
- Results used to determine the conditions for the definitive study: There was a dose response at the rates of 0.1, 1.0, 10, 100 and 1000 mg Supragil WP/kg dry soil tested in the range finder test. However, the dose response to nitrate production is not a normal rate progression in that, at 1000 mg/kg the nitrification step of the nitrogen transformation pathway in the soil has been affected and there is a build-up of ammonium, hence the value for nitrate produced at Day 28 is less than that of the 100 mg/kg rate. However, addition of the products of mineralisation of the organic nitrogen, that is, the summation of the ammonium-N and nitrate-N, shows increasing production of mineral nitrogen up to 1000 mg/kg.
The EC50 for nitrate concentration at Day 28 estimated at 801 mg/kg Supragil WP and the EC50 for nitrate production from Day 0-28 was estimated at 550 mg/kg Supragil WP in the range finder test.
Nominal and measured concentrations:
Range Finder Test:
Water Control
0.10, 1.00, 10.0, 100 and 1000 mg Supragil WP/kg dry soil
Definitive Test:
Water Control
1.00, 10.0, 100, 500 and 1000 mg Supragil WP/kg dry soil
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
164 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC25
Effect conc.:
260 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
420 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
See table 1 and 2

During the 0-28 day period the soil nitrate transformation rates (mg/kg dry soil/day) in the soil treated at concentrations of 1.0, 10.0, 100, 500 and 1000 mg Supragil WP/kg dry soil varied by 4.7%, 16.0%, 89.3%, -52.2%, and -104%, respectively, from the mean of the water control group. At the lower treatment levels there is stimulation of organic nitrogen breakdown resulting in increased levels of nitrate formed on the 1.00, 10.0 and 100 mg Supragil WP/kg dry soil, with no increase in the ammonium concentration as organic N is being mineralised to ammonium and then by nitrification to nitrate. In the higher treatment groups, 500 and 1000 mg Supragil WP/kg dry soil there is a build up of ammonium in the soil at Day 28 and demonstrates that the nitrification process cannot convert all the ammonium to nitrate, resulting in the lower levels of nitrate production.
The nitrite results have not been reported as nitrite-N levels were all below the limit of detection of 0.5 mg/L. This is as expected in an aerobic soil system, however, due to the chemistry of the reaction that determines nitrate, it is incorrect to report the TON value as nitrate unless the absence of nitrite has been confirmed.

Supragil WP at the rates tested did affect soil pH values during the course of the study as the build up of ammonium in the 500 and 1000 mg/kg treatment rate has increased the pH in these groups on Day 28.
Results with reference substance (positive control):
none
Reported statistics and error estimates:
none

Table 1: Effects on Organic Nitrogen Transformation in Soil after Treatment with Supragil WP:

Treatment

Day 28 data

Day 0 to Day 28 rate data

Mean Ammonium

(mg/kg dry soil)

% Variation (ammonium production) from control

Mean Nitrate (mg/kg dry soil)

% Variation (Nitrate) from control

Mean Nitrate Production

(mg/kg/day)

% Variation (from control)

Deionised water control

0.72

NA

286.7

NA

5.97

NA

1.00 mg Supragil WP/kg

0.68

-6.09

294.9

2.85

6.25

4.66

10.0 mg Supragil WP/kg

0.72

0.00

319.1

11.3*

6.92

16.0

100 mg Supragil WP/kg

0.86

18.3

452.7

57.9*

11.30

89.3*

500 mg Supragil WP/kg

92.2

12624

215.6

-24.8*

2.85

-52.2*

1000 mg Supragil WP/kg

114.7

15735

118.3

-58.7*

-0.25

-104.3*

All concentrations on a dry soil basis

* Statistically different to control (Dunnett’s Test, two-tail, p ≤ 0.05)

Some values are calculated using un-rounded values

There is an overall stimulation of organic nitrogen breakdown in the 10 mg Supragil WP/kg dry soil rate, which is reflected in the results by increased levels of the end product of mineralisation and nitrification, nitrate, by Day 28. At higher rates of treated soil, there is still an increase in the rate of organic N breakdown but the nitrification process (ammonium conversion to nitrate) is slowed and hence there is a build up of ammonium that has not been converted to nitrate at the higher rates.

Table 2: Statistical Analysis Summary Nitrogen Transformation to Nitrate in Soil after Treatment with Supragil WP:

 

 

EC10

mg Supragil WP/kg

EC25

mg Supragil WP/kg

EC50

mg Supragil WP/kg

Day 0-28 Nitrate production rate

164

260

420

Day 28 Nitrate concentration

210

376

739

Validity criteria fulfilled:
yes
Remarks:
The variation in nitrate concentration in replicate water control samples varied by -1.4 to 1.6 % at Day 28, which was no greater than +/-15 %.
Conclusions:
Supragil WP (sodium diisopropylnaphthalenesulphonate) tested at 1.00, 10.0, 100, 500 and 1000 mg/kg dry soil, showed a statistically significant effect on soil nitrogen mineralisation compared to the control treatment at 100, 500 and 1000 mg product/kg dry soil on the rate of transformation at the end of the 28 day test period (Dunnett’s two tail, p=0.05). The concentration of nitrate in the soil at the end of the test period was statistically different from the control treatment in all treatements except the lowest at 1.00 mg prduct/kg.
For the Day 0 to 28 period, the EC10 , EC25 and EC50 for the rate of nitrate production (net mineralisation) were estimated at 164, 260 and 420 mg Supragil WP/kg dry soil, respectively.
As there is a difference in the rate of nitrate production between the control replicates and the higher treatment rates was greater than 25% from Day 0 to Day 28, Supragil WP can be assessed as having a long-term influence on the nitrogen transformation in soils at concentrations ≥100 mg product/kg dry soil.
Executive summary:

This study determined the effects of sodium diisopropylnaphthalenesulphonate, herein referred to as Supragil WP, on the nitrogen transformation activity in a LUFA standard soil type 2.3, according to procedures described in OECD Guideline 216 (2000).

 

The soil used in the study had a sand content of 59.7 %, a pH of 6.8 (measured in water), with an organic carbon content of 0.67 % and a microbial biomass of 198 mg Cmic/kg which was 2.96 % of the total organic carbon. The soil met the guideline requirement of not less than 1% biomass carbon as % total organic carbon. In most cases, a soil with these characteristics represents a worst-case situation since adsorption of the test chemical to soil is minimal and consequently its availability to the microflora is maximal. As a result of a range finder test conducted at 0.1,1.00, 10.0, 100 and 1000 mg Supragil WP mg/kg dry soil , the soil was amended with powdered lucerne and the soil treated with test item at nominal concentrations of 1.00, 10.0, 100, 500 and 1000 mg Supragil WP/kg dry soil. Each treatment group and a deionised water control was replicated three times. The products of the process of mineralisation were extracted from the soil on Days 0 and 28 after treatment and incubation at 20oC ± 2oC.The extracted solutions were analysed for nitrate, nitrite and ammonium nitrogen concentrations from the samples taken on Day 0 and Day 28 and the results converted to mg/kg dry soil basis.

 

Supragil WP (sodium diisopropylnaphthalenesulphonate) tested at 1.00, 10.0, 100, 500 and 1000 mg/kg dry soil, showed a statistically significant effect on soil nitrogen mineralisation compared to the control treatment at 100, 500 and 1000 mg product/kg dry soil on the rate of transformation at the end of the 28 day test period (Dunnett’s two tail, p=0.05). The concentration of nitrate in the soil at the end of the test period was statistically different from the control treatment in all treatements except the lowest at 1.00 mg product/kg.

 

For the Day 0 to 28 period, the EC10, EC25 and EC50for the rate of nitrate production (net mineralisation) were estimated at 164, 260 and 420 mg Supragil WP/kg dry soil, respectively.

 

As there is a difference in the rate of nitrate production between the control replicates and the higher treatment rates was greater than 25% from Day 0 to Day 28, Supragil WP can be assessed as having a long-term influence on the nitrogen transformation in soils at concentrations ≤100 mg product/kg dry soil.

Description of key information

Supragil WP (sodium diisopropylnaphthalenesulphonate) tested at 1.00, 10.0, 100, 500 and 1000 mg/kg dry soil, showed a statistically significant effect on soil nitrogen mineralisation compared to the control treatment at 100, 500 and 1000 mg product/kg dry soil on the rate of transformation at the end of the 28 day test period (Dunnett’s two tail, p=0.05). The concentration of nitrate in the soil at the end of the test period was statistically different from the control treatment in all treatements except the lowest at 1.00 mg product/kg. For the Day 0 to 28 period, the EC10 , EC25 and EC50 for the rate of nitrate production (net mineralisation) were estimated at 164, 260 and 420 mg Supragil WP/kg dry soil, respectively. As there is a difference in the rate of nitrate production between the control replicates and the higher treatment rates was greater than 25% from Day 0 to Day 28, Supragil WP can be assessed as having a long-term influence on the nitrogen transformation in soils at concentrations ≥100 mg product/kg dry soil.

For the Day 0 to 28 period, the EC10 , EC25 and EC50 of Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda for the rate of nitrate production (net mineralisation) were thus determined by analogy (Read across approach) to be 164, 260 and 420 mg/kg dry soil, respectively.

For the Day 0 to 28 period, the EC10 , EC25 and EC50 of Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda for the rate of nitrate production (net mineralisation) were thus determined by analogy (Read across approach) to be 164, 260 and 420 mg/kg dry soil, respectively.

Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda

Key value for chemical safety assessment

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

Additional information

The toxicity to soil microorganisms of Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda is determined by a read-across approach based on analogue substance Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda.

A GLP-compliant study (C. Hamwijk, 2016), scored as Klimisch 2 and flagged as a key study, is available on the Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda.

The following results were determined:

EC10

mg Supragil WP/kg

EC25

mg Supragil WP/kg

EC50

mg Supragil WP/kg

Day 0-28 Nitrate production rate

164

260

420

Day 28 Nitrate concentration

210

376

739