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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
- Concentrations:0-10-32-100-32-1000 mg Cd/kg dry soil
Vehicle:
no
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20°C+- 2°C
Moisture:
treatment: 0-10-32-100-320-1000 mg/kg dry soil
Moisture day 28 (% w/w):14.2-14.4-14.2-14.1-14.2-14.0
Details on test conditions:
The study was carried out with a single, common agricultural soil as described in OECD Guideline 217 (2000). The soil used wasLUFA standard soil type 2.3(Batch number F2.35111) and was supplied byLUFA-Speyer, Obere Langgasse 40, 67346 Speyer, Germany.
The soil had a sand content of 63.7%, a pH of 7.1 (measured in water), with an organic carbon content of 0.94% and a microbial biomass of not less than 1% of the total organic carbon content (actual value 3.25%). 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.
The soil sampling was carried out according to ISO 10381-6 (1993) as recommended in OECD 217 (2000). Details of the soil collection, site history and soil treatment and soil characterisation data (see Appendices 2 and 3) were provided by LUFA-Speyer. Soil was received at CEMAS on the 11 January 2012 and was stored in a refrigerator nominally at 4°C, until required.

Acclimation of the soil used for the study was started on 20 January 2012, where 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 ± 2oC for 6 days before Day 0. The MWHC of the soil was declared at 35.6%.
The microbial biomass of this batch of soil was determined in accordance with ISO 14240-1 (1997) and CEMAS SOP CEM-3370 Determination of microbial biomass carbon by substrate induced respiration. The microbial biomass was 3.25% of the total organic carbon.
Each replicate was contained within a 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. Three replicates were prepared for the control treatment (deionised water), and the test item treatments. Each replicate contain 500 g (dry weight equivalent) of soil.
The test soil was amended (mixed) with glucose at each sampling time point to elicit a maximum respiratory response. The amount of glucose required for this soil was pre-determined (when the biomass was measured) as 1.0 mg glucose/g dry weight of soil.
A satellite moisture control vessel was set up for each test for the sole purpose of monitoring moisture content. Moisture content of the soil was maintained at 40% ( 5%) of the MWHC and at Days 7, 14 and 28 the weight of the moisture control vessel was determined as a guide to test vessel water loss. Weight loss since the previous sampling occasion was restored by the addition of deionised water to all replicates in all treatments, based on the assumption that all vessels had experienced similar moisture change.
Soil was taken from one replicate from each treatment for pH (water) determination at the start and end of the Study. Soil pH was determined using SOP CEM-3373 Determination of the pH of water, soil and sediment samples in water and/or salt solutions (0.01MCaCl2, 0.1M KCl, 1.0M KCl).
Soil was taken from one replicate from each treatment for moisture and dry matter content determination at the end of the Study, using SOP CEM-30
A series of approximately 1500 g (dry weight equivalent) amounts of soils were treated with the appropriate test item treated sand and a volume of deionised water to bring the moisture of the soil to approximately 42% MWHC required for the test duration. The soil was mixed thoroughly using a mixer and then equally divided into the three replicate vessels for each treatment.
As soon as possible after dosing approximately 100 g (dry weight equivalent) samples were taken from each replicate for the determination of glucose-induced respiration rates in soil respiration chambers. Further sub-samples were taken after 28 days.
The sub-samples were mixed with an optimum level of glucose for the soil to stimulate respiration. The glucose was mixed with talc (magnesium silicate) as a carrier. The carbon dioxide produced over a 12 hour period was then determined using an infrared carbon dioxide analyser and automatic column switching unit (GHU) (ADC 2250 and GHU, Analytical Development Company, Great Amwell, Herts, UK). Methodology followed SOP CEM-3370 Determination of Microbial Biomass Carbon by Substrate Induced Respiration.
Carbon dioxide (CO2) evolution from each sample was determined using the following procedure and equipment:
Soil respiration chambers: Stainless steel tubes (respirometers) approximately 50 mm internal diameter by 250 mm in length with bungs at each end and a suitable stopper at the air inlet. Additional equipment included a carbon dioxide scrubber, gas flow meter and flow control valves. The respiration chambers were connected in parallel to a source of CO2 free air, a gas handling unit and then to an infrared gas analyser (IGA). Each channel received a nominal flow of CO2–free air at ca.100 mL/minute, throughout the 12 hour period.
Gas Handling Unit (GHU): The gas multiplexing unit sequentially diverted the air exiting a number of different soil respiration chambers (channels) to a common Gas Analyser, which was set to switch between channels (respiration chambers) every five minutes.
The Infrared Gas Analyser (IGA): ADC (Analytical Development Company, Great Amwell, Herts, UK): Type 2250 carbon dioxide analyser. Transfers the CO2 measurements to a computer and the concentration of CO2 in the air exiting the channel being sampled determined every minute. Recordings during the five minute period up to the final reading were used to confirm that the CO2 concentration had reached a steady level for that channel.
At five minute intervals the glucose and talc mixture was added to the soil samples, well mixed and then added to the soil respiration chambers. Following a period of five to ten minutes from adding the soil and glucose to the first channel (channel 1) logging of the CO2 concentration in the air flowing from channel 1 was started. The Gas Handling Unit was set to switch between channels automatically every 5 minutes as 12 channels were used.In this way each channel was measured once per hour for a minimum of 12 hours.
Soil respiration rates as CO2 concentrations measured for each specimen over a twelve hour period were calculated by using the CO2 readings as CO2 in mg/m3 and converted to CO2 mg/kg dry weight soil/hour using the following equation:-

mg CO2/kg/hour = (CO2 in mg/m3 x flow rate in L/hour/1000) x 10

(where 1000 is the factor to convert m3 to litres, and 10 is the factor to convert the amount CO2 produced from the 100 g soil sample to that produced per 1 kg of soil).

The average rate of CO2 production over each 12 hour period was calculated for each replicate.
The glucose induced respiration rate was determined as the average rate of CO2 production over each 12 hour period and was calculated for each replicate. CO2 concentrations were calculated by converting CO2 readings from CO2 in mg/m3 to CO2 in mg/kg dry soil/hour.
Nominal and measured concentrations:
Nominal concentration: 0-10-100-320-1000 mg Cd/kg DW
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 g/kg soil dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
Cd
Basis for effect:
respiration rate
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 2.1 g/kg soil dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
CdTe
Basis for effect:
respiration rate
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
2.1 g/kg soil dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
respiration rate
Reported statistics and error estimates:
The mean carbon dioxide production rate was calculated for each treatment and the percent deviation from the control calculated. Statistical analysis was performed (using ToxCalc, 1999) to determine significant treatment related effects compared to the control.

By Day 28, microbial carbon respiration in soil treated with Cadmium telluride at10, 32, 100, 320 and1000 mg Cd/kg dry soilvaried by +3.43, +5.66, +2.34, +3.77 and +1.99% respectively from the mean of the control group. The percentage variation within the control samples for carbon dioxideproduction (mg CO2/kg/hr) ranged from -2.7% to +1.8% on Day 28. This data fulfils the validity criterion that the variation between control replicates from the average should not be greater than 15%.

Treatment (mg Cd/kg dry soil)

Mean Carbon dioxide

(mg/kg/hr)

% Difference from Control

Control (Deionised water)

21.60

n/a

10

22.34

3.43

32

22.83

5.66

100

22.11

2.34

320

22.42

3.77

1000

22.03

1.99

Soil pH (measured in water)

 

Treatment (mg Cd/kg dry soil)

Day 0

Day 28

Control (deionised water)

7.3

7.4

10

7.4

7.4

32

7.4

7.4

100

7.5

7.5

320

7.5

7.5

1000

7.5

7.5

Targeted Initial Moisture Level

The MWHC of the soil was declared at 35.6% (see Appendix 2). Soil moisture levels for the study were targeted at 40% (±5%) of the MWHC of the soil. Achieved values are given below:

Dry Matter and Moisture Levels at Day 28 (Study completion)

 

Treatment (mg Cd/kg dry soil)

Dry Matter

Day 28a

(% w/w)

Moisture Day 28a

(% w/w)

Achieved % MWHCb

Control (deionised water)

85.8

14.2

39.9

10

85.6

14.4

40.4

32

85.8

14.2

39.9

100

85.9

14.1

39.6

320

85.8

14.2

39.9

1000

86.0

14.0

39.3

aTaken from replicate 1 vessel from each treatment.

bMoisture content divided by the MWHC of the soil, multiplied by 100.

 

Validity criteria fulfilled:
yes
Conclusions:
Test done according to standard protocol.
Executive summary:

Statistical analysis showed there was no significant difference (p>0.05) between any treatment rate of cadmium/kg dry soil and the control treatment for soil carbon transformations at Day 28 (Dunnett’s two-tail test, ToxCalc version 5.0.23, 1999). The EC50 was determined as >1000 mg Cd/kg dry soil.

Description of key information

No acute effects were seen up to a concentration of 2.1 g CdTe/kg DW or 1 g Cd/kg DW. 
As Cd2+ ion determines ecotoxicity, the PNEC deriviation will be based on the Cd 2+ ion as determined in the RA (ECB 2007).

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
2 100 mg/kg soil dw

Additional information

Statistical analysis showed there was no significant difference (p>0.05) between any treatment rate of Cd/kg dry soil and the control treatment for soil carbon transformations at day 28. So, no effects were seen up to a concentration of 2.1 g CdTe/kg DW or 1 g Cd/kg DW. As Cd2+ ion determines ecotoxicity, the PNEC deriviation will be based on the Cd 2+ ion as determined in the RA (ECB 2007).