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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 09 November 2018 and 14 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
deviations did not impact on the validity of the study (see any other information on materials and methods)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
see comments above
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a WAF of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide
Batch: Y180510A
Purity: 95.0%
Physical State/Appearance: Black powder
Expiry Date: 31 May 2019
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and 100 mg/L loading rate WAF test group from the test preparation at 0 hours and from the pooled replicates (R1 to R4) at 48 hours for immediate quantitative analysis of the silicon content.
Duplicate sets of samples of the control and 100 mg/L loading rate WAF test group were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Following the definitive test, a trial was conducted to estimate the background level of silicon present in the dilution medium after it had been stirred for 95 hours under test conditions. Samples from this experiment were taken before the stirring in a glass container, before siphoning through glass wool and after siphoning through glass wool. Samples were analyzed immediately.
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and complex nature of the test item for the purposes of the study the test medium was prepared as a WAF of the test item.

Test water:
Reconstituted water (ISO media) used for both the range-finding and definitive tests. ISO media was used to reduce the possibility of chelation of the components of the test item with the test media.

Validation of Mixing Period:
Preliminary investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of dissolved test item in the WAF. A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in deionized 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 the stirring periods the WAFs were allowed to settle for 1-Hour prior to removal of the aqueous phase. Visual observations indicated that a significant amount of dispersed test item was present. As such the dispersed fraction was removed from the aqueous fraction by filtration through a glass wool plug (24-Hour prep) or a glass wool plug and one sheet of filter paper (96-Hour prep). After filtration through one glass wool plug and one sheet of filter paper the 24-Hour preparation was examined under the microscope; no micro-dispersions of test item were observed to be present. Samples taken for chemical analysis gave the following results:
The results are summarized as follows:

Nominal Loading Rate: 100 mg/L
24 Hours Measured Concentration (mg/l Si): 0.0450
96 Hours Measured Concentration (mg/l Si): 1.22

It is evident from this work that increasing the stirring period significantly increased the silicon concentration in the WAF between the 24-Hour and 96-Hour preparation periods and so the WAF was prepared using a 95-Hour stirring period followed by a 1-Hour standing period.

In a trial conducted after the definitive test, analysis of dilution medium taken before and after stirring showed that there was a background level of silicon present in the medium and this increased after stirring following contact with the borosilicate glassware and glass wool. Consequently, the control data from the definitive test was used to indicate the background level of silicon in the dilution medium. The preliminary investigational work therefore may have also detected levels of silicon due to borosilicate glassware and glass wool but this was not acknowledged at the time.

Range-finding Test:
The loading rate to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.
Nominal amounts of test item (5, 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 respectively. 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 95 hours and the mixtures allowed to stand for 1-Hour. Microscopic observations made on the 100 mg/L loading rate WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the dispersed material by filtering the WAF 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 dispersed material in the WAF removed by mid-depth siphoning (the first 75 to 100 mL passed through the filter was discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs. Microscopic observations of the 100 mg/L loading rate WAF was performed after filtering and showed that undissolved test item was still visible. Further filtration was performed using filter paper. The test preparations were all clear colorless solutions.

A sample of each loading rate WAF was taken for chemical analysis at 0 hours and the measured concentration of silicon determined as an indicator of the presence of test item. All samples were stored frozen prior to analysis. No sample was taken at 48 hours for the 100 mg/L loading rate WAF due to the test solution being discarded accidentally.

Definitive Test:
Based on the results of the range-finding test a "limit test" was conducted at a loading rate of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines no immobilization or adverse reactions to exposure were observed.

A nominal amount of test item (500 mg) was added to the surface of 5 liters of test water to give the 100 mg/L loading rate. 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 95 hours and the mixture allowed to stand for 1-Hour. Observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the dispersed material by filtering the WAF 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 dispersed material in the WAF removed by mid-depth siphoning (the first 75 to 100 mL passed through the filter was discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed no micro-dispersions to be present no further filtration was required.

The concentration of silicon in the test preparations were verified by chemical analysis at 0 and 48 hours

















Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnids were maintained in 150 mL glass vessels 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). 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
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
Test temperature:
Temperature was maintained at approximately 21 °C throughout the test.
pH:
The reconstituted water had a pH of 7.8 ±0.2 adjusted (if necessary) with NaOH or HCl.
Dissolved oxygen:
8.70 to 9.00 mg O2/L
Nominal and measured concentrations:
Range-finding Test:
Nominal: 1.0, 10, 100 mg/L WAF
Measured: Chemical analysis of the fresh test preparations at 0 hours showed measured silicon concentrations to range from 0.081 to 0.36 mg/L silicon. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 0.23 to 0.35 mg/L for the 1.0 mg/L and 10 mg/L loading rate WAF only.

Definitve Test:
Nominal: 100 mg/L WAF
Measured: Chemical analysis of the fresh (0 hours) and aged test preparations (48 hours) showed measured silicon concentrations in the 100 mg/L loading rate WAF to be 0.22 mg/L silicon and 0.20 mg/L silicon, respectively.

Details on test conditions:
Range-Finding Test:
In the range-finding test five daphnids were placed randomly in each test and control vessel, containing the test preparations, maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C. There was 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 numbers of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.

Definitive Test - Exposure Conditions:
As in the range-finding test 150 mL glass vessels containing approximately 100 mL of test preparation were used. At the start of the test five daphnids were placed at random in each test and control vessels, containing the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C. There was a photoperiod of 16 hours light (between 200 and 1200 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period.

Assessments:
Test Organism Observations:
Any immobilization 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 immobilized if they were unable to swim within 15 seconds after gentle agitation.

Water Quality Criteria:
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.

Vortex Depth Measurements:
The vortex depth was recorded at the start and end of the mixing period.










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:
48 h
Dose descriptor:
NOELR
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:
No immobilization or sub-lethal effects of exposure were observed throughout the test at the loading rates of 1.0, 10 and 100 mg/L WAFs employed.

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 immobilization or adverse reactions to exposure were observed.

Chemical analysis of the fresh test preparations at 0 hours showed measured silicon concentrations to range from 0.081 to 0.36 mg/L silicon. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 0.23 to 0.35 mg/L for the 1.0 mg/L and 10 mg/L loading rate WAF only (in error, no sample was taken at 100 mg/L loading rate WAF). As no control media samples were analyzed, the results have not been corrected for background levels of silicon.

Definitive Test:
Chemical Analysis of Test Loading Rates:
Chemical analysis of the fresh (0 hours) and aged test preparations (48 hours) showed measured silicon concentrations in the 100 mg/L loading rate WAF to be 0.22 mg/L silicon and 0.20 mg/L silicon, respectively. Silicon was also measured in the control samples. At 0 hours a measured silicon concentration of 0.15 mg/L was determined; analysis of the duplicate sample confirmed the presence of silicon (0.078 mg silicon /L). At 48 hours the control sample had a measured silicon concentration of 0.19 mg/L.

In a trial conducted after the definitive test, analysis of dilution medium taken before and after stirring showed that there was a background level of silicon present in the medium; 0.061 mg silicon/L before stirring and this increased to 0.23 mg silicon/L after stirring following contact with the borosilicate glassware and glass wool. Consequently, the control data from the definitive test was used to indicate the background level of silicon in the dilution medium, which was subtracted from the silicon levels measured in the 100 mg/L loading rate WAF, to give estimates of the concentrations of silicon attributable to test item. The silicon concentration after correction for the control (using the original value) was 0.070 mg/L at 0 hours and 0.0040 mg/L at 48 hours in the 100 mg/L loading rate WAF.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on the nominal loading rate.

Immobilization Data:
The silicon present, whether from test item or background levels, did not cause any adverse effects as there was no immobilization in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours. Inspection of the immobilization data gave the following results:

Time Point: 24 Hours
EL50: >100 mg/L Loading Rate WAF
NOEL: 100 mg/L Loading Rate WAF

Time Point: 48 Hours
EL50: >100 mg/L Loading Rate WAF
NOEL: 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:
No sub-lethal effects of exposure were observed throughout the test.

Validation Criteria:
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress 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.

Water Quality Criteria:
Temperature was maintained at approximately 21 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Throughout the test the light intensity was observed to be in the range 783 to 882 lux.

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 Item 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 preparation was observed to have a clear colorless water column with test item floating at the water surface and dispersed throughout. After 95 hours stirring and a 1-Hour standing period the 100 mg/L preparation was observed to have test item floating at the water surface and dispersed throughout. Visual observation showed test item dispersed throughout the water column and therefore it was considered justifiable to remove the dispersed material by filtering the WAF through a glass wool plug (2 to 4 cm in length). Microscopic examination of the WAF after filtration showed there to be no micro-dispersions of test item present.

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.




















Results with reference substance (positive control):
A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. The positive control was conducted between 25 September 2018 to 27 September 2018.

Exposure conditions for the positive control were similar to those in the definitive test with an exception for the light intensity. In the definitive test at 0 and 48 hours the light intensity measured was in excess of the range specified in the study plan (500 to 1000 lux), the maximum light intensity recorded was 1037 lux. Given that the recorded light intensity was only marginally in excess of the specified range and as the daphnids in the control group did not show any mortalities or sub-lethal effects, this deviation was considered not to have adversely affected the results of the test.

Analysis of the immobilization data was carried out using the Weibull analysis using linear maximum-likelihood regression at 24 and 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: 1.1 mg/L (95% Confidence Limits 0.95 - 1.3 mg/L)
NOEC: 0.56 mg/L
LOEC: 1.0 mg/L

48 Hours:
EC50: 0.8 mg/L (95% Confidence Limits 0.12 - 1.5 mg/L)
NOEC: 0.56 mg/L
LOEC: 1.0 mg/L

A level of 20% immobilization was recorded in the 0.32 mg/L test concentration; however, as the immobilization recorded in the 0.56 mg/L test concentration was only 10% immobilization it was considered unlikely that the effect observed was as a result of exposure to the reference item and therefore the NOEC was 0.56 mg/L.

The NOEC is based upon equal to or less than 10% immobilization at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal Loading Rate (mg/L)

Observations (Initial Population: 5 Per Replicate)

24 Hours

48 Hours

Cumulative Immobilized Daphnia

Observations

Cumulative Immobilized Daphnia

Observations

R1

R2

R1

R2

R1

R2

R1

R2

Control

0

0

5 N

5 N

0

0

5 N

5 N

1.0

0

0

5 N

5 N

0

0

5 N

5 N

10

0

0

5 N

5 N

0

0

5 N

5 N

100

0

0

5 N

5 N

0

0

5 N

5 N

R = Replicate

N = Normal

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Loading Rate (mg/L

24 Hours

Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

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 Immobilized Daphnia (Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

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

Water Quality Measurements

Nominal Loading Rate (mg/L)

0 Hours

24 Hours

48 Hours

pH

Mg O2/L

Temperature °C

Temperature °C

pH

Mg O2/L

Temperature °C

Control

R1

7.9

8.70

21

20.8

7.9

9.00

21

100

R2

7.9

8.80

21

20.8

7.9

8.93

21

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item gave the following results:
48 Hours EL50 (mg/L Loading Rate WAF): >100
48 Hours NOEL (mg/L Loading Rate WAF): 100

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

Introduction:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods:

Due to the low aqueous solubility and complex nature of the test item, 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 WAF of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of approximately 21°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Since the test item was multi-component, verification of the test item concentrations was based on the analysis of the silicon component as an indicator of the presence of the test item but does not represent the test item concentration as a whole.

Results:

Chemical analysis of the fresh (0 hours) and aged test preparations (48 hours) showed measured silicon concentrations in the 100 mg/L loading rate WAF to be 0.22 mg/L silicon and 0.20 mg/L silicon, respectively. Silicon was also measured in the control samples, with levels of 0.15 and 0.19 mg/L silicon at 0 and 48 hours, respectively. In a trial conducted after the definitive test, analysis of dilution medium taken before and after stirring showed that there was a background level of silicon present in the medium, of 0.061 mg/L silicon before stirring and this increased to 0.23 mg/L after stirring following contact with the borosilicate glassware and glass wool. Consequently, the control data from the definitive test was used to indicate the background level of silicon in the dilution medium, which was subtracted from the silicon levels measured in the 100 mg/L loading rate WAF, to give estimates of the concentrations of silicon attributable to test item. The silicon concentration after correction for the control was 0.070 mg/L silicon at 0 hours and 0.0040 mg/L silicon at 48 hours in the 100 mg/L loading rate WAF.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on the nominal loading rate.

Exposure of Daphnia magna to the test item gave the following results:

Time Point (Hours)

EL50 (mg/L Loading Rate WAF)

NOEL (mg/L)

48

>100

100

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

Description of key information

Introduction:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods:

Due to the low aqueous solubility and complex nature of the test item, 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 WAF of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of approximately 21°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Since the test item was multi-component, verification of the test item concentrations was based on the analysis of the silicon component as an indicator of the presence of the test item but does not represent the test item concentration as a whole.

Results:

Chemical analysis of the fresh (0 hours) and aged test preparations (48 hours) showed measured silicon concentrations in the 100 mg/L loading rate WAF to be 0.22 mg/L silicon and 0.20 mg/L silicon, respectively. Silicon was also measured in the control samples, with levels of 0.15 and 0.19 mg/L silicon at 0 and 48 hours, respectively. In a trial conducted after the definitive test, analysis of dilution medium taken before and after stirring showed that there was a background level of silicon present in the medium, of 0.061 mg/L silicon before stirring and this increased to 0.23 mg/L after stirring following contact with the borosilicate glassware and glass wool. Consequently, the control data from the definitive test was used to indicate the background level of silicon in the dilution medium, which was subtracted from the silicon levels measured in the 100 mg/L loading rate WAF, to give estimates of the concentrations of silicon attributable to test item. The silicon concentration after correction for the control was 0.070 mg/L silicon at 0 hours and 0.0040 mg/L silicon at 48 hours in the 100 mg/L loading rate WAF.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on the nominal loading rate.

Exposure ofDaphnia magnato the test item gave the following results:

Time Point (Hours)

EL50 (mg/L Loading Rate WAF)

NOEL (mg/L)

48

>100

100

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

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information