Fatty acids, tall-oil, compds. with ethanolamine

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:

27 September 2016 to 25 November 2016

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)

Deviations:

yes

Remarks:

test temperature deviation with no impact on results or integrity of study (see below)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

test temperature deviation with no impact on results or integrity of study (see below)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC-MS using an external standard

Details on sampling:

RANGE FINDING TEST
- A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions.
- All samples were stored frozen prior to analysis.

DEFINITIVE TEST
- Samples were taken from the control and from the freshly prepared bulk test preparation of each surviving test group at 0 and 24 hours.
- Samples were also taken from the pooled replicates (replicates R1-R4) at 24 and 48 hours.
- Samples were stored frozen prior to analysis.

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST SYSTEM
- The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Annex 2) 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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

Theoretical hardness of 250 mg/L as CaCO3 (see Annex 4, attached)

Test temperature:

21 to 22 °C (see Table 3, attached)

pH:

7.7 to 8.0 (see Table 3, attached)

Dissolved oxygen:

8.1 to 8.9 mf O2/L (see Table 3, attached)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

RANGE FINDING TEST
- nominal loading rates of 1.0, 10 and 100 mg/L

INITIAL EXPERIMENTS
- Nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L.

DEFINITIVE TEST
- Nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

TEST WATER
- Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined in Annex 4 (attached).

EXPERIMENTAL DESIGN AND STUDY CONDUCT
- 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 Water Accommodated Fraction (WAF) of the test item.

VALIDATION OF MIXING PERIOD
- Preliminary work (see Annex 5, attached) was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

RANGE-FINDING TEST
- The loading rates to be used in the definitive test were 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 (5.0, 50 and 500 mg) were each separately added to a glass slide and suspended in the water column of 5 L 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 23 hours and the mixtures allowed to stand for 1 hour. Visual 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 (2-4 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 75-100 ml discarded). After filtration through a glass wool plug, the test media was observed to be cloudy, as a result, it was considered necessary to filter again through filter paper to give the 1.0, 10 and 100 mg/L loading rate WAFs. Observations at the start of the test showed the media to be a clear colourless solution.
- In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 22 to 23 °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. Although the temperatures recorded were greater than those stated in the study plan, this was considered not to have had an impact on the outcome of the test. Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised daphnids were recorded.
- The control group was maintained under identical conditions but not exposed to the test item.

INITIAL EXPERIMENTS
- Prior to the definitive test, initial experiments were conducted at nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L.
- For each of the initial experiments, nominal amounts of test item (5.0, 9.0, 16, 28 and 50 mg) were each separately added to a glass slide and suspended in the water column of 5 L of test water to give the 1.0, 1.8, 3.2, 5.6 and 10 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 23 hours and the mixtures allowed to stand for 1 hour. Visual 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 (2-4 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 75-100 mL discarded) to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed that no undissolved test item was present.
- Test conditions were similar to those described for the definitive test. However, in all of the initial experiments, inconsistent immobilisation patterns were observed. Due to the paste like nature of the test item, it was considered possible that an uneven layer was being applied to the glass slides for each loading rate which would result in variable dissolution/dispersion into the water column. A more refined technique was therefore considered necessary whereby the test item was spread in a much thinner layer on the surface of the glass slide to ensure that the maximum surface area was obtained.

DEFINITIVE TEST
- Based on the results of the initial experiments, the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.
- Nominal amounts of test item (5.0, 16, 50, 160 and 500 mg) were each separately added in a thin layer to a glass slide and then suspended in the water column of 5 L of test water close to the base of the beaker to give the 1.0, 3.2, 10, 32 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 23 hours and the mixtures allowed to stand for 1 hour. Visual 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 (2-4 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 75-100 mL discarded). After siphoning, whilst, the 1.0, 3.2 and 10 mg/L loading rates were observed to have no micro dispersions of test item remaining, the 32 and 100 mg/L loading rates were still observed to be cloudy. As a result, it was considered appropriate to filter the 32 and 100 mg/L loading rates twice through filter paper. After this further filtration, both the 32 and 100 mg/L loading rates were observed to be slightly cloudy however, it was considered that further filtration would not be successful at removing any more test item.
- All loading rates were prepared in the same way for the media renewal at 24 hours, however, the 10, 32 and 100 mg/L loading rates were not used as all daphnids had been immobilised by this point.
- The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24 and 48 hours (see Annex 6, attached).

EXPOSURE CONDITIONS
- In the definitive test, 150 mL glass jars containing approximately 100 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in 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 with 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.
- Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media. Concentrations at which 100% immobilisation was observed after 24 hours exposure were not renewed.

TEST ORGANISM OBSERVATIONS
- 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 for approximately 15 seconds after gentle agitation.
- Microscopic observations were conducted on any immobilised daphnia in the 10, 32 and 100 mg/L loading rates due to the presence of dispersed test item in the media.

WATER QUALITY CRITERIA
- Water temperature, pH and dissolved oxygen were recorded daily throughout the test.
- Measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations.
- The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter.
- 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.

VALIDATION CRITERIA
- Results of the test are considered valid if the following performance criteria are met:
(a) No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).
(b) The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.

MAJOR COMPUTERISED SYSTEMS
- Statistical analysis: ToxRat
- Building management: Delta Control System

Reference substance (positive control):

yes

Remarks:

potassium dichromate (investigation conducted between 29 February 2016 and 02 March 2016; see Annex 3, attached)

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

3.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: based on loading rate

Remarks:

confidence limits not determined

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TEST
- Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (attached).
- No immobilization was observed at 1.0 mg/L loading rate WAF, however, immobilisation was observed at 10 and 100 mg/L loading rate WAF. Microscopic observation of the immobilized daphnia showed that no microparticles were adhered to the daphnids.
- Based on this information loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L were selected for the initial experiments.
- Chemical analysis of the test preparations at 0 hours (see Annex 6, attached) showed measured test concentrations of between 0.26 and 6.1 mg/L were obtained. Chemical analysis of the test preparations at 48 hours showed that measured concentrations of between less than the limit of quantification (LOQ) of the analytical method, determined to be 0.049 mg/L, and 5.8 mg/L were obtained, suggesting that the test item was potentially unstable under test conditions.

INITIAL EXPERIMENTS
- Inspection of the data from the initial experiments suggested that as the test item was being added in a lump to a glass slide and suspended in the water column, it was being removed from the slide and into the water column at different rates/in different amounts in each loading rate. The way that these lumps were then being dispersed in the water column was resulting in amounts of bio-available test item in each loading rate not being concentration specific.
- As a result, for the purposes of the definitive test, the test item was to be smeared onto the glass slides in a thin layer, allowing for a much more even dispersion of the test item into the water column. In addition, as a precautionary measure, a wider concentration range was employed.

DEFINITIVE TEST
- Chemical analysis (see Annex 6) of the freshly prepared 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF test preparations at 0 hours showed measured test concentrations to range from 0.44 to 11 mg/L, and of the corresponding old or expired test preparations at 24 hours to range from 0.21 to 4.6 mg/L.
- Chemical analysis of the freshly prepared 1.0 and 3.2 mg/L loading rate test preparations at 24 hours showed that measured concentrations of 0.29 and 0.80 mg/L were obtained respectively and of the corresponding old or expired test preparations at 48 hours showed that measured concentrations of 0.14 and 0.38 mg/L were obtained respectively.
- The dissolved test item may have been one or several components of the test item. 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 nominal loading rates only.

IMOBILISATION DATA
- Cumulative immobilisation data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (attached).
- The relationship between percentage immobilisation and concentration at 48 hours is given in Figure 1 (attached).
- Analysis of the immobilisation data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the nominal loading rates gave the results shown in the table below.
- The No Observed Effect Loading rate after 24 and 48 hours exposure was 1.0 mg/L loading rate WAF. Correspondingly the Lowest Effect Loading rate was considered to be 3.2 mg/L loading rate WAF.
- The slopes and their standard error of the response curve at 24 and 48 hours was 11.3 (SE = 0.44).

SUB-LETHAL EFFECTS
- Sub-lethal effects of exposure were observed in the 3.2 mg/L loading rate WAF group. This response was a pale colour (see Table 2, attached).
- As the test item was observed to form a dispersion in the test diluents at the higher loading rates, microscopic inspection of the immobilised daphnids was performed. This examination revealed that no particles of test item had adhered to the daphnids indicating physical toxicity was not a factor.

VALIDATION CRITERIA
- The test was considered to be valid given that no more than 10% 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
- The results of the water quality measurements are given in Table 3 (attached).
- Temperature was maintained at 21 to 22 °C throughout the test.
- There were no treatment related differences for oxygen concentration or pH.

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 W AFs.
- At the start of both the first and second mixing periods the 1.0, 3.2, 10, 32 and 100 mg/L loading rates were observed to be clear water columns with test item suspended in a thin layer on a glass slide suspended close to the base of the beaker.
- During the first stirring period, after 23 hours stirring and a 1-Hour standing period the 1.0 and 3.2 mg/L loading rates were observed to be clear colourless water columns with test item floating throughout, the 10 mg/L loading rate was observed to be a clear colourless water column with a substantial amount of test item floating throughout, the 32 mg/L loading rate was observed to be a cloudy water column with test item floating throughout and the 100 mg/L loading rate was observed to be a very cloudy water column with test item floating in chunks throughout.
- During the second stirring period, after 23 hours stirring and a 1-Hour standing period, the 1.0, 3.2 and 10 mg/L loading rates were observed to be clear colourless water columns with small amounts of test item floating throughout, the 32 mg/L loading rate was observed to be a slightly cloudy water column with larger lumps of test item floating throughout and the 100 mg/L loading rate was observed to be a very cloudy water column. Visual examination of the WAFs after both stirring periods showed that dispersed test item was present and therefore it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length).
- Microscopic examination after filtering showed the glass wool plug had removed all of the dispersed test item in the 1.0, 3.2 and 10 mg/L loading rates, however, further filtration through filter paper was required for the 32 and 100 mg/L loading rates. After further filtration, the 32 and 100 mg/L loading rates were still observed to be cloudy, however, it was considered that any further filtration would be unsuccessful at removing any more of the dispersed material.
- At the start and throughout the test all control and 1.0, 3.2 and 10 mg/L loading rates were observed to be clear colourless solutions. At 0 hours, the 32 and 100 mg/L loading rates were observed to be slightly hazy dispersions, whilst at 24 hours, the 32 mg/L loading rate was observed to be a very slightly hazy dispersion with test item settled on the bottom and the 100 mg/L loading rate was observed to be a slightly hazy dispersion with test item settled on the bottom.

Results with reference substance (positive control):

- The results from the positive control with potassium dichromate were within the normal range for this reference item (see Annex 3, attached).

Reported statistics and error estimates:

STATISTICAL ANALYSIS
- The EL50 values at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression.
- The Lowest Observed Effect Loading Rate and the No Observed Effect Loading Rate at 24 and 48 hours were calculated using the Step-down Cochran Armitage Procedure.
- All results were calculated using the ToxRat Professional computer software package (TOXRAT).

RESULTS FROM PROBIT ANALYSIS OF IMOBILISATION DATA

Time (h)	EL50 (mg/L loading rate WAF)	95 % confidence limits (mg/L loading rate WAF)
24	3.7	Not determined
48	3.7	Not determined

Validity criteria fulfilled:

yes

Conclusions:

Following exposure of the freshwater invertebrate, Daphnia magna to the test item, the EL50 (48h) value was reported as 3.7 mg/L loading rate WAF (confidence limits not determined). The No Observed Effect Loading Rate (NOEL) was reported as 1.0 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) reported as 3.2 mg/L loading rate WAF.

Executive summary:

GUIDELINE

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,"Daphniasp., 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, and initial experiments, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for 48 hours at a temperature of 21 to 22 °C under semi - static test conditions. The number of immobilised daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

RESULTS

Chemical analysis of the fresh 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF test preparations at 0 hours showed measured test concentrations to range from 0.44 to 11 mg/L, and of the corresponding old or expired test preparations at 24 hours to range from 0.21 to 4.6 mg/L. Chemical analysis of the freshly prepared 1.0 and 3.2 mg/L loading rate WAF test preparations at 24 hours showed that measured concentrations of 0.29 and 0.80 mg/L were obtained respectively and of the corresponding old or expired test preparations at 48 hours showed that measured concentrations of 0.14 and 0.38 mg/L were obtained respectively. The dissolved test item may have been one or several components of the test item. 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 nominal loading rates only.

 

CONCLUSION

Following exposure of the freshwater invertebrate, Daphnia magna, to the test item, the EL50 (48h) value was reported as 3.7 mg/L loading rate WAF (confidence limits not determined). The No Observed Effect Loading Rate (NOEL) was reported as 1.0 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) reported as 3.2 mg/L loading rate WAF.

Description of key information

Following exposure of the freshwater invertebrate, Daphnia magna to the test item, the EL50 (48h) value was reported as 3.7 mg/L loading rate WAF (confidence limits not determined). The No Observed Effect Loading Rate (NOEL) was reported as 1.0 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) reported as 3.2 mg/L loading rate WAF (OECD 202 and EU Method C.2).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.7 mg/L

Additional information

GUIDELINE

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,"Daphniasp., 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, and initial experiments, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L for 48 hours at a temperature of 21 to 22 °C under semi - static test conditions. The number of immobilised daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

RESULTS

Chemical analysis of the fresh 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF test preparations at 0 hours showed measured test concentrations to range from 0.44 to 11 mg/L, and of the corresponding old or expired test preparations at 24 hours to range from 0.21 to 4.6 mg/L. Chemical analysis of the freshly prepared 1.0 and 3.2 mg/L loading rate WAF test preparations at 24 hours showed that measured concentrations of 0.29 and 0.80 mg/L were obtained respectively and of the corresponding old or expired test preparations at 48 hours showed that measured concentrations of 0.14 and 0.38 mg/L were obtained respectively. The dissolved test item may have been one or several components of the test item. 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 nominal loading rates only.

 

CONCLUSION

Following exposure of the freshwater invertebrate, Daphnia magna, to the test item, the EL50 (48h) value was reported as 3.7 mg/L loading rate WAF (confidence limits not determined). The No Observed Effect Loading Rate (NOEL) was reported as 1.0 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) reported as 3.2 mg/L loading rate WAF.