Young daphnids (in house breeding at B.T.), not older than 24 hours at the start of the test, are exposed to a range of concentrations of the test substance in the test medium (ISO water) for 48 hours in static or semistatic conditions. The exposure is carried out under controlled conditions of temperature and light cycle. The immobilization is measured at 24 and 48 hours and compared with the control.

TEST SYSTEM
Daphnia magna
A toxic reference substance is tested twice a year to check the sensitivity of the organisms.

STUDY DESIGN
Range finding test includes at least 5 concentrations of the test substance and 1 untreated control (4 replicates/concentration or control) with 5 daphnids/replicate
Limit test is carried out with 1 concentration (100 mg/L or at solubility limit) of the test substance and 1 untreated control (4 replicates/concentration or control) with 5 daphnids/replicate
Dose-response test includes at least 5 concentrations of the test substance and
1 untreated control (4 replicates/concentration or control) with 5 daphnids/replicate

Analytical checks, to verify the exposure concentrations in the medium, are performed  for static test at time 0 and at time 48 hours on control and on each test substance sample and, for semi-static test at time 0, time 24 hours and time 48 hours on control and on each test substance sample.

The analysis are performed using a method validated according to SANCO/825/00 rev. 8.1 and SANCO/3029/99 rev. 4 guidance documents.

ENDPOINTS
Immobilization: EC₅₀ at 48 hours as the main endpoint and as other endpoints: EC₁₀, EC₂₀, LOEC/NOEC.
Study includes GLP management and reporting.

REFERENCES AND GUIDELINES
OECD Guideline for Testing of Chemicals, No. 202 (13^th April 2004) – Daphnia sp., acute immobilization test.
SANCO/825/00 rev. 8.1 (16/11/2010) – Guidance document on pesticide residue analytical methods.
SANCO/3029/99 rev. 4 (11/07/2000) – Residues: guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (Part A, section 4) and Annex III (Part A, section 5) of Directive 91/414.

Young female Daphnia (the parent animals, in house breeding at B.T.), aged less than 24 hours at the start of the test, will be exposed at determinate concentrations of test substance  in the test medium (M4) for a period of 21 days in semi-static conditions.
At the end of the test, the reproductive output of the parent animals will be evaluated based on the total number of living offspring produced.

TEST SYSTEM
Daphnia magna
A toxic reference substance is tested twice a year to check the sensitivity of the organisms.

STUDY DESIGN
Range finding for acute immobilization test is carried out with at least 5 concentrations of the test substance and 1 untreated control (4 replicates/concentration or control)  with 5 daphnids/replicate
Range finding for reproduction test includes at least 5 concentrations of the test substance and 1 untreated control (10 replicates/concentration or control) with 1 daphnid/replicate
Limit test is carried out with 1 concentration (as 10 mg/L or 100 mg/L) of the test substance and 1 untreated control (10 replicates/concentration or control) with 1 daphnid/replicate
Dose-response test includes at least 5 concentrations of the test substance and
1 untreated control (10 replicates/concentration or control) with 1 daphnid/replicate

Analytical checks, to verify the exposure concentrations in the medium, are performed at time 0, 3 times per week  (in spent and fresh solutions) and at 21 days on control and on each test substance sample.

The analysis are performed using a method validated according to SANCO/825/00 rev. 8.1 and SANCO/3029/99 rev. 4 guidance documents.

ENDPOINTS
Reproduction: EC₁₀, EC₂₀, EC₅₀ and LOEC/NOEC.
Study includes GLP management and reporting.

REFERENCES AND GUIDELINES
OECD Guideline fo Testing of Chemicals, No. 211 (2^nd October 2012) – Daphnia magna reproduction test.
OECD Series on Pesticides, Number 67: “OECD guidance to the environmental safety evaluation of microbial biocontrol agents” (17^th February 2012).
Environmental Canada, EPS 1/RM/44: “Guidance Document for Testing the Pathogenicity and Toxicity of New Microbial Substances to Aquatic and Terrestrial Organisms” (March 2004).
SANCO/825/00 rev. 8.1 (16/11/2010) – Guidance document on pesticide residue analytical methods.
SANCO/3029/99 rev. 4 (11/07/2000) – Residues: guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (Part A, section 4) and Annex III (Part A, section 5) of Directive 91/414.

First instar Chironomus riparius larvae (in house breeding at BT) are exposed to a range of concentrations of the test substance in the test medium (M4 medium) for a period of 48 hours. The exposure is carried out under controlled conditions of temperature and light cycle. The immobilization is recorded at 24 and 48 hours and compared with the control.

TEST SYSTEM
Chironomus riparius 
A toxic reference substance is tested twice a year to check the sensitivity of the organisms.

STUDY DESIGN
Range finding test includes at least 5 concentrations of the test substance and 1 untreated control (2 or 4 replicates/concentration or control) with 5 larves/replicate
Limit test is carried out with 1 concentration (100 mg/L or at solubility limit) of the test substance and 1 untreated control (4 replicates/concentration or control) with 5 larves/replicate
Dose-response test includes at least 5 concentrations of the test substance and
1 untreated control (4 replicates/concentration or control) with 5 larves/replicate

Analytical checks, to verify the exposure concentrations in the medium, are performed at time 0 and at time 48 hours on control and on each test substance sample.

The analysis are performed using a method validated according to SANCO/825/00 rev. 8.1 and SANCO/3029/99 rev. 4 guidance documents.

ENDPOINTS
Immobilization: EC₅₀ at 48 hours as the main endpoint and as other endpoints: EC₁₀, EC₂₀, LOEC/NOEC.
Study includes GLP management and reporting.

REFERENCES AND GUIDELINES
OECD Guideline fo Testing of Chemicals, No. 235 (28^th July 2011) – Chironomus sp., acute immobilization test.
SANCO/825/00 rev. 8.1 (16/11/2010) – Guidance document on pesticide residue analytical methods.
SANCO/3029/99 rev. 4 (11/07/2000) – Residues: guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (Part A, section 4) and Annex III (Part A, section 5) of Directive 91/414.

First instar Chironomus riparius larvae (in house breeding at BT) are exposed to a concentration range of the test substance in sediment/water system, for 28 days. The test substance is spiked into the sediment and first instar larvae are subsequently introduced into test beakers in which the sediment and water concentrations have been stabilised. The exposure is carried out under controlled conditions of temperature and light cycle. Chironomid emergence and development rate is measured at the end of the test and compared with the control. Larval survival and weight may also be measured after 10 days if required (using additional replicates as appropriate).

TEST SYSTEM
Chironomus riparius 
A toxic reference substance is tested twice a year to check the sensitivity of the organisms.

STUDY DESIGN
Range finding test includes at least 5 concentrations of the test substance and 1 untreated control (1 replicate/concentration or control) with 20 larves/replicate
Limit test is carried out with 1 concentration (1000 mg/Kg) of the test substance and 1 untreated control (6replicates/concentration or control) with 20 larves/replicate
Dose-response test includes at least 5 concentrations of the test substance and
1 untreated control (4 replicates/concentration or control) with 20 larves/replicate

Analytical checks, to verify the exposure concentrations (where relevant or required samples include water, pore water and sediment), are performed at time 0 and at time 28 days on control and on each test substance sample.

The analysis are performed using a method validated according to SANCO/825/00 rev. 8.1 and SANCO/3029/99 rev. 4 guidance documents.

ENDPOINTS
Emergence, development rate: EC₅₀ at 28 days as the main endpoint and as other endpoints: EC₁₅, LOEC/NOEC.
Study includes GLP management and reporting.

REFERENCES AND GUIDELINES
OECD Guideline fo Testing of Chemicals, No. 218 (13^th April 2004) – Sediment-water Chironomid toxicity test using spiked sediment.
SANCO/825/00 rev. 8.1 (16/11/2010) – Guidance document on pesticide residue analytical methods.
SANCO/3029/99 rev. 4 (11/07/2000) – Residues: guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (Part A, section 4) and Annex III (Part A, section 5) of Directive 91/414.

First instar Chironomus riparius larvae (in house breeding at BT) are exposed to a concentration range of the test substance in sediment/water system, for 28 days. The test starts by placing first instar larvae into the test beakers containing the sediment/water system and subsequently spiking the test substance into the water. The exposure is carried out under controlled conditions of temperature and light cycle. Chironomid emergence and development rate is measured at the end of the test. Larval survival and weight may also be measured after 10 days if required (using additional replicates as appropriate).

TEST SYSTEM
Chironomus riparius 
A toxic reference substance is tested twice a year to check the sensitivity of the organisms.

STUDY DESIGN
Range finding test includes at least 5 concentrations of the test substance and 1 untreated control (1 replicate/concentration or control) with 20 larves/replicate
Limit test is carried out with 1 concentration of the test substance and 1 untreated control (6 replicates/concentration or control) with 20 larves/replicate
Dose-response test includes at least 5 concentrations of the test substance and
1 untreated control (4 replicates/concentration or control) with 20 larves/replicate

Analytical checks, to verify the exposure concentrations (where relevant or required samples include water, pore water and sediment), are performed at time 0 and at time 28 days on control and on each test substance sample.

The analysis are performed using a method validated according to SANCO/825/00 rev. 8.1 and SANCO/3029/99 rev. 4 guidance documents.

ENDPOINTS
Emergence, development rate: EC₅₀ at 28 days as the main endpoint and as other endpoints: EC₁₅, LOEC/NOEC.
Study includes GLP management and reporting.

REFERENCES AND GUIDELINES
OECD Guideline fo Testing of Chemicals, No. 219 (13^th April 2004) – Sediment-water Chironomid toxicity test using spiked water.
SANCO/825/00 rev. 8.1 (16/11/2010) – Guidance document on pesticide residue analytical methods.
SANCO/3029/99 rev. 4 (11/07/2000) – Residues: guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (Part A, section 4) and Annex III (Part A, section 5) of Directive 91/414.

The fishes are exposed to the test substance for 96 hours under static or semi-static conditions. Temperature is controlled and from 12 to 16 hours photoperiod applied. Mortalities are recorded at 24, 48, 72 and 96 hours and the concentration which kill 50% of the fishes determined.

TEST SYSTEM
Oncorhynchus mykiss
A toxic reference substance is tested twice a year to check the sensitivity of the organisms.

STUDY DESIGN
Range finding test includes at least 5 concentrations of the test substance and 1 untreated control (7 replicates/concentration or control) with 1 fish/replicate.
Limit test is carried out with 1 concentration (100 mg/L or at solubility limit) of the test substance and 1 untreated control (7 replicates/concentration or control) with 1 fish/replicate.
Dose-response test includes at least 5 concentrations of the test substance and 1 untreated control (7 replicates/concentration or control) with 1 fish/replicate.

Analytical checks, to verify the exposure concentrations in the medium, are performed  for static test at time 0 and at time 96 hours on control and on each test substance sample.
For semi-static test the analysis are carried out at time 0, 24 hours, 48 hours, 72 hours (in spent and fresh solutions) and at 96 hours on control and on each test substance sample.

The analysis are performed using a method validated according to SANCO/825/00 rev. 8.1 and SANCO/3029/99 rev. 4 guidance documents.

ENDPOINTS
Mortality: LC₅₀ at 96 hours as the main endpoint and as other endpoints: LC₁₀, LC₂₀, LOEC/NOEC.
Study includes GLP management and reporting.

REFERENCES AND GUIDELINES
OECD Guideline fo Testing of Chemicals, No. 203 (17^th July 1992) – Fish, acute toxicity test.
SANCO/825/00 rev. 8.1 (16/11/2010) – Guidance document on pesticide residue analytical methods.
SANCO/3029/99 rev. 4 (11/07/2000) – Residues: guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (Part A, section 4) and Annex III (Part A, section 5) of Directive 91/414.