Soybean meal, protein extn. residue

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 April 2018 to 03 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Samples were taken from the control and the 100 mg/L loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates (R1 to R4) at 48 hours for quantitative analysis. All samples were analysed on the day of sampling.
- Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

SOLUBILITY OF THE TEST MATERIAL
- Due to the low aqueous solubility and complex nature of the test material, for the purposes of the study the test medium was prepared as a WAF of the test material.
- Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances, in the WAF.
- A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in deionised reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1 Hour standing period the mixtures were then removed by filtration through a glass wool plug and one sheet of filter paper to remove as much dispersed test material as present. Observations made on the preparations following filtration showed slightly hazy dispersions were formed. It was considered that further filtration at this point would not have removed any more of the dispersed test material present.
- It was evident from this work that increasing the stirring period did not increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.

EXPERIMENTAL PREPARATION
- A nominal amount of test material (200 mg) was added to the surface of 2 litres of test water to give the 100 mg/L loading rate. After the addition of the test material, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. Microscopic observations made on the WAF indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid depth siphoning (the first 75 to 100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF was performed after filtering which showed no microdispersions of the test item to be present, however, the preparation was observed to be a cloudy dispersion and therefore the WAF was also filtered through filter paper.

VORTEX DEPTH MEASUREMENT
The vortex depth was recorded at the start and end of the mixing period.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: First instar
- Source: Derived from in-house laboratory cultures
- Age: Less than 24 hours old
- Feeding during test: No

ACCLIMATION
- Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C.
- The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

20 - 21 °C

pH:

7.5 - 7.9

Dissolved oxygen:

6.8 - 8.8 mg O2/L

Nominal and measured concentrations:

Nominal: 100 mg/L loading rate WAF
Measured: 0 hours: 38 mg C/L, 48 hours: 31 mg C/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL beakers
- Type: Closed. The test vessels were then covered to reduce evaporation.
- Material, size, headspace, fill volume: 100 mL
- Renewal rate of test solution (frequency/flow rate): Not renewed
- Aeration: The test vessels were not aerated.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water – Elendt M7 Medium: H3BO3 0.715 mg/L, MnCl2.4H2O 0.090 mg/L, LiCl 0.077 mg/L, RbCl 0.018 mg/L, SrCl2.6H2O 0.038 mg/L, NaBr 0.004 mg/L, Na2MoO4.2H2O 0.016 mg/L, CuCl2.2H2O 0.004 mg/L, ZnCl2 0.013 mg/L, CoCl2.6H2O 0.010 mg/L, KI 0.0033 mg/L, Na2SeO3 0.0022 mg/L, NH4VO3 0.00058 mg/L, Na2EDTA.2H2O 0.625 mg/L, FeSO4.7H2O 0.249 mg/L, CaCl2.2H2O 293.8 mg/L, NaHCO3 64.8 mg/L, MgSO4.7H2O 123.3 mg/L, Na2SiO3.9H2O 10 mg/L, KCl 5.8 mg/L, NaNO3 0.274 mg/L, K2HPO4 0.184 mg/L, KH2PO4 0.143 mg/L, Thiamine hydrochloride 0.075 mg/L, Cyanocobalamine (vitamin B12) 0.0010 mg/L and D(+) biotin (vitamin H) 0.00075 mg/L. The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 °C.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The light intensity during the light period was measured using an ATP Instrumentation Lux meter. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Light intensity: 865 to 968 lux

EFFECT PARAMETERS MEASURED:
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilised if they were unable to swim within 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L loading rate WAF
- Nominal amounts of test item (5.0, 50 and 500 mg) were each separately added to the surface of 5 liters of test water to give the 1.0, 10 and 100 mg/L loading rates. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering which showed no microdispersions of the test item to be present, however, the 100 mg/L preparation was observed to be a cloudy dispersion and therefore the WAFs were also filtered through filter paper. Following filtration through filter paper, the 1.0 and 10 mg/L preparations were clear and colorless whereas the 100 mg/L preparation was a cloudy dispersion.
- In the range-finding test five daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised daphnids was recorded.
- The control group was maintained under identical conditions but not exposed to the test item.
- Samples were taken at 0 and 48 hours for TOC analysis.
- Results used to determine the conditions for the definitive study: Yes. In the range-finding test after 48 hours, no immobilisation was observed at the 10 and 100 mg/L loading rate WAFs, however, all daphnia at the 1.0 mg/L loading rate WAF were immobilised. Given that the TOC results indicated that the test material was correctly dosed and as there was no immobilisation in the 10 and 100 mg/L loading rate WAFs, the immobilisation at the 1.0 mg/L loading rate WAF was considered to be attributable to unknown contamination. No sub lethal effects of exposure were observed throughout the test. Based on this information a single loading rate of four replicates of 100 mg/L, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilisation or adverse reactions to exposure were observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

other: NOEL

Effect conc.:

100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST
- In the range-finding test after 48 hours, no immobilisation was observed at the 10 and 100 mg/L loading rate WAFs, however, all daphnia at the 1.0 mg/L loading rate WAF were immobilised.
- TOC analysis of the 1.0, 10 and 100 mg/L loading rate WAF test preparations at 0 hours showed measured concentrations of less than the LOQ, 4.7 and 40 mg C/L (LOQ considered to be 1.0 mg C/L). TOC analysis of the 1.0, 10 and 100 mg/L loading rate WAF test preparations at 48 hours showed measured concentrations of less than the 1.6, 3.9 and 35 mg C/L.
- Given that the TOC results indicated that the test material was correctly dosed and as there was no immobilisation in the 10 and 100 mg/L loading rate WAFs, the immobilisation at the 1.0 mg/L loading rate WAF was considered to be attributable to unknown contamination.
- No sub lethal effects of exposure were observed throughout the test.
- Based on this information a single loading rate of four replicates of 100 mg/L, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilisation or adverse reactions to exposure were observed.


DEFINITIVE TEST
Chemical Analysis of Test Loading Rates
- Total Organic Carbon (TOC) analysis of the 100 mg/L loading rate WAF test preparation at 0 hours showed the measured concentration was 38 mg C/L. A slight decline in TOC concentration was observed at 48 hours to 31 mg C/L.
- The dissolved test material may have been one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

Immobilisation Data
- There was one immobilised daphnid observed in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours.
- Inspection of the immobilisation data gave the following results:
24 h EL50 = >100 mg/L loading rate WAF
48 h EL50 = >100 mg/L loading rate WAF
- The NOEL rate after 24 and 48 hours exposure was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Sub-Lethal Effects
- A sub lethal effect of exposure was observed in the 100 mg/L loading rate WAF group. This response was pale coloration.

Vortex Depth Measurements
- The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.

Observations on Test Material Solubility
- Observations on the test media were carried out during the mixing and testing of the WAF.
- At the start of the mixing period the 100 mg/L loading rate was observed to be clear colourless water column with test material floating on water surface. After 23 hours stirring and a 1 Hour standing period the 100 mg/L loading rate was observed to remain as at start of mixing. Microscopic examination of the WAF showed microdispersions observed and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). Microscopic observations of the WAF performed after filtering showed no microdispersions of the test material to be present, however, the preparation was observed to be a cloudy dispersion and therefore the WAF was also filtered through filter paper. Examination after filtering using a glass wool plug and filter paper showed the preparation remained a cloudy dispersion.
- During the test the control was observed to be a clear, colourless solution and the 100 mg/L loading rate was observed to be very slightly cloudy dispersion.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilisation data was carried out using the Trimmed Spearman-Karber method at 24 hours and the Binomial Distribution method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:
24 hours: EC50 = 0.79 mg/L (95 % CL: 0.73 - 0.86 mg/L), NOEC = 0.56 mg/L and LOEC = 1.0 mg/L
48 hours: EC50 = 0.75 mg/L (95 % CL: 0.56 - 1.0 mg/L), NOEC = 0.56 mg/L and LOEC = 1.0 mg/L
The NOEC is based upon equal to or less than 10 % immobilisation at this concentration.
The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24-Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L. The results from the positive control with potassium dichromate were within the normal range for the reference material.

Reported statistics and error estimates:

An estimate of the EL10, EL20 and EL50 values was given by inspection of the immobilisation data.

Validation Criteria

- The test was conside red to be valid given that less than 10 % of the control daphnids showed immobilisation or other signs of disease or stress (actual: 5 %) and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels (actual: ≥6.8 mg/L).

Table 1: Total Organic Carbon Analysis Results from the Range Finding Test

Samples	Nominal Loading Rate (mg/L)	Concentration of TOC (mg C/L)
0 Hours	1.0	<LOQ
	10	4.712
	100	40.37
48 Hours	1.0	1.578
	10	3.949
	100	34.80

LOQ = Limit of quantification which was considered to be 1.0 mg C/L

Table 2: Total Organic Carbon Analysis Results from the Definitive Test

Samples	Nominal Loading Rate (mg/L)	Concentration of TOC (mg C/L)	Concentration of TOC Corrected for Control (mg C/L)
0 Hours	Control	2.251	-
	100	40.23	37.98
48 Hours	Control	<LOQ	-
	100	30.52	30.52

LOQ = Limit of quantification which was considered to be 1.0 mg C/L

Table 3: Cumulative Immobilisation Data and Observations in the Definitive Test

Nominal Loading Rate (mg/L)	24 Hours
	Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N
Nominal Loading Rate (mg/L)	48 Hours
	Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	1	0	0	0	1	5	4 N	5 N	5 N	5 N
100	1	0	0	0	1	5	4 P	5 P	5 P	5 P

Rep = Replicate

N = Normal

P = Pale

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test material gave a 48 Hour EL10, EL20 and EL50 value of greater than 100 mg/L loading rate WAF. The NOEL rate was 100 mg/L loading rate WAF.

Executive summary:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions.

Due to the low aqueous solubility and complex nature of the test material, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range finding test, 20 daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test material at a nominal loading rate of 100 mg/L for 48 hours at a temperature of approximately 20 to 21 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Total Organic Carbon (TOC) analysis of the 100 mg/L loading rate WAF test preparation at 0 hours showed the measured concentration was 38 mg C/L. A slight decline in TOC concentration was observed at 48 hours to 31 mg C/L.

The dissolved test material may have been one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Under the conditions of this study, the test material gave a 48 Hour EL10, EL20 and EL50 value of greater than 100 mg/L loading rate WAF. The NOEL rate was 100 mg/L loading rate WAF.

Description of key information

Under the conditions of this study, the test material gave a 48 Hour EL10, EL20 and EL50 value of greater than 100 mg/L loading rate WAF. The NOEL rate was 100 mg/L loading rate WAF.

Additional information

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Due to the low aqueous solubility and complex nature of the test material, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range finding test, 20 daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test material at a nominal loading rate of 100 mg/L for 48 hours at a temperature of approximately 20 to 21 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Total Organic Carbon (TOC) analysis of the 100 mg/L loading rate WAF test preparation at 0 hours showed the measured concentration was 38 mg C/L. A slight decline in TOC concentration was observed at 48 hours to 31 mg C/L.

The dissolved test material may have been one or several components of the test material. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test material as a whole, the results were based on nominal loading rates only.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Under the conditions of this study, the test material gave a 48 Hour EL10, EL20 and EL50 value of greater than 100 mg/L loading rate WAF. The NOEL rate was 100 mg/L loading rate WAF.