[No public or meaningful name is available]

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-08-2020 to 08-10-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

OECD TG 203 ; adopted June 2019

Deviations:

no

Remarks:

No positive control conducted as part of the study

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Remarks:

No positive control conducted as part of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: November 2019 ; signature: August 2020

Analytical monitoring:

yes

Details on sampling:

- Concentrations: the saturated solution (in the limit test item loading rate concentration) and the control were analytically verified via GC-MS/MS in fresh media at the start of exposure (0 hours) and at one renewal interval (0 and 72 hours) in corresponding old media at 24 hours and 96 hours (the end of the exposure). For the limit test item loading rate concentration (100 mg/L nominal) : the measured concentration of the test item was 0.163 μg/L at the test start (0 hours), 0.110 μg/L in old media (24 hours), 41.1 μg/L in fresh media (72 hours) at renewal and 28.6 μg/L in old media at test end (96 hours). The control concentrations were < LOQ at all time points. Equivalent Geometric mean measured concentrations were determined: 0 (control) and 2.14 μg/L, which were based on analysis during the definitive test period. The analytical values indicate that the sampling points were not completely homogeneous, which can be caused by the low water solubility of the test item, which is in accordance with the results of related studies (Water Solubility and/or Acute Daphnia Toxicity – cited in the full study report). For further comments about the observed concentrations, see below.
- Sampling method: Water samples were taken from the control and all test group(s) at 0 and 72 hours (fresh media/renewal); and 24, 96 hours (old media) for quantitative analysis. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: All original samples were stored at room temperature (ca. 20 ± 2 °C) before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item exposures was prepared from stock solution. A saturated solution prepared with of 100 mg/L of the test item was tested in a limit test. The limit concentration was selected based upon the very low solubility of the test item and in accordance with the OECD TG 203 Guidelines for testing. Additionally, the selection of the test concentration is based on the derivation of a threshold concentration (TC) from the data of the test item of results of a previous daphnia acute immobilization test (EC50 (0-48 h) = > saturated solution (nominal concentration of 100 mg/L) and of a previous alga toxicity test (ErC50 (0-72 h) = >saturated solution (nominal concentration of 100 mg/L). Related study citations provided in the full study report. A saturated solution with a nominal loading of 100 mg/L of the test saturated solution item was prepared with dilution water one day before the start of the exposure (at day -1) and one day prior each renewal of the test solutions (at day 0, 1 and 2). A glass bottle was filled with an appropriate amount of the dilution water. The headspace in the glass flask was minimized. An appropriate volume of the test item was weighed out and placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 24 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour at room temperature for separation of undissolved test item. Thereafter, the clear water phase was removed by siphoning from the bottom of the glass flask. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative. The saturated solution was used as limit concentration. Presence of undissolved test item during preparation and during the test was not observed. The saturated solution was used as a limit concentration. For the negative control : Dilution water without test item incubated under the same conditions as the test groups was treated equivalent to the preparation of the saturated solution, above.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: A negative/blank control without test item or reference item was also included.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Prior to start of the exposure, the test solutions were checked for undissolved test item. Presence of undissolved test item during preparation and during the test was not observed.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish (Brachydanio rerio or Danio rerio)
- Strain: Not reported. Although full details on the brood recognised supplier are recorded in the full study report.
- Source: Recognised supplier (recorded in full study report). All fish used in the test were obtained from a single brood stock.
- Age at study initiation (mean and range, SD): Not reported. All fish had 9 days of acclimatization and mortality < 5% within these days prior to the start of the exposure.
- Length at study initiation (length definition, mean, range and SD): The fish density in the test vessels was 0.096 g fish per litre test solution. Average body length at the test end: 1.41 cm. Average body weight at the test end: 0.034 g. In the treated/control groups the loading was ca. 0.096 g/L (i.e. < 1.0 g fish/litre) based on the above information.
- Method of breeding: Not reported.
- Maintenance of the brood fish: Not reported. See information below for acclimatisation and exposure.

ACCLIMATION
- Acclimation period: 9 days (i.e. > 48 hours)
- Acclimation conditions (same as test or not): Holding at test facility at 23 ± 2 °C and diffuse light (540 – 1000 lux, photoperiod of 12 to 16 h light / 12 to 8 h dark). The water was changed at least once per week. The dissolved oxygen concentration was more than 80% of the air saturation value. Holding water: tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine. Nominal water parameters: Total hardness: 40 - 250 mg CaCO3/L ; pH-value: 6.0 - 8.5 ; Oxygen saturation: ≥ 80 % of air saturation value and/or (recent measurements): Acidity: 0.2 mmol/L ; Alkalinity: 0.5 mmol/L ; Conductivity: 157 μS/cm
- Type and amount of food during acclimation: Standard food (recognised supplier)given to satiation (4% of the fish body weight) per feeding day.
- Feeding frequency during acclimation: Feeding was conducted during acclimation (provided 3 times per week. Food was given to satiation (4% of the fish body weight) per feeding day. Until 24-hours prior to test initiation.
- Health during acclimation (any mortality observed): None reported (< 5% during 9 days pre-test).

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

FEEDING DURING TEST
- Food type: Not applicable. Feeding was conducted during acclimation (provided 3 times per week. Food was given to satiation (4% of the fish body weight) per feeding day. Until 24-hours prior to test initiation.
- Amount: No feeding during exposure. Not applicable.
- Frequency: No feeding during exposure. Not applicable.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Remarks on exposure duration:

According to OECD TG 203 guidelines.

Post exposure observation period:

None.

Hardness:

ca. 62 mg/L (expressed as CaCO3) measured on day 0 from dilution water (TOC = < 2 mg/L)
The water used to create the dilution water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine. Nominal water parameters: Total hardness: 40 - 250 mg CaCO3/L ; pH-value: 6.0 - 8.5 ; Oxygen saturation: ≥ 80 % of air saturation value and/or (recent measurements): Acidity: 0.2 mmol/L ; Alkalinity: 0.5 mmol/L ; Conductivity: 157 μS/cm

Test temperature:

21 - 25 °C, controlled at ± 1 °C (actual: 22.1°C to 22.8°C and mean 22.4 °C)

pH:

7.62-7.81 (fresh media); 7.49-7.70 (expired media)

Dissolved oxygen:

94-100% ASV (fresh media); 81-90% (expired media)

Salinity:

Not applicable.

Conductivity:

The water used to create the dilution water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine. Nominal water parameters: Total hardness: 40 - 250 mg CaCO3/L ; pH-value: 6.0 - 8.5 ; Oxygen saturation: ≥ 80 % of air saturation value and/or (recent measurements): Acidity: 0.2 mmol/L ; Alkalinity: 0.5 mmol/L ; Conductivity: 157 μS/cm

Nominal and measured concentrations:

Concentrations: 0 mg/L (control) and saturated loading rate (limit test) 100 mg/L nominal
Equivalent Geometric mean measured concentrations were determined: 0 (control) and 2.14 μg/L, based on analysis during the definitive test period.
- Other: For the limit test item loading rate concentration (100 mg/L nominal) : the measured concentration of the test item was 0.163 μg/L at the test start (0 hours), 0.110 μg/L in old media (24 hours), 41.1 μg/L in fresh media (72 hours) at renewal and 28.6 μg/L in old media at test end (96 hours). The control concentrations were < LOQ at all time points. Equivalent Geometric mean measured concentrations were determined: 0 (control) and 2.14 μg/L, which were based on analysis during the definitive test period. The analytical values indicate that the sampling points were not completely homogeneous, which can be caused by the low water solubility of the test item, which is in accordance with the results of related studies (Water Solubility and/or Acute Daphnia Toxicity – cited in the full study report). Test item did not need to be corrected for recoveries as procedural recoveries (% recovery calculated to weighed amount of the test item) were within the range of 90% to 109% and with a mean 102%, during exposure period.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria of 3 L were used (dimensions: 11.5 x 13 x 20 cm, depth of water: approx. 17 cm) and covered with a glass plate.
- Type (delete if not applicable): closed (covered with a glass plate)
- Material, size, headspace, fill volume: 2.5 L fill volume (0.5 L headspace)
- Aeration: No (dissolved oxygen: not less than 60% of air saturation value during the exposure period)
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): A semi-static test with daily renewal of the test media was performed.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): One (1)
- No. of vessels per control (replicates): One (1)
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: In the treated/control groups the loading was ca. 0.096 g/L (i.e. < 1.0 g fish/litre)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared in house ; the water used to create the dilution water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine. Nominal water parameters: Total hardness: 40 - 250 mg CaCO3/L ; pH-value: 6.0 - 8.5 ; Oxygen saturation: ≥ 80 % of air saturation value and/or (recent measurements): Acidity: 0.2 mmol/L ; Alkalinity: 0.5 mmol/L ; Conductivity: 157 μS/cm
- Total organic carbon: TOC = < 2 mg/L ; measured on day 0 from dilution water
- Particulate matter: Not reported.
- Metals: total hardness: ca. 62 mg/L (expressed as CaCO3) measured on day 0 from dilution water and conductivity: 157 μS/cm
- Pesticides: Not reported. Nitrates: 6.87 mg/L measured on day 0 from dilution wate for days 0 to 4
- Chlorine: See above.
- Alkalinity: See above.
- Ca/mg ratio: See above.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: The pH-value, temperature and oxygen saturation were measured in all test vessels at the start and the end of exposure as well as daily and after renewal of the test media from fresh and old test media. Chlorine and nitrate were measured from the batch of the dilution water used in the test. Total hardness and TOC were determined at the start of the exposure in the dilution water. During the test, the water temperature was recorded continuously (once per hour) with a data logger. The light intensity on the surface of the test aquaria was measured at the start of the exposure. The solution appearance and behaviour was observed daily before and after renewal.

OTHER TEST CONDITIONS
- Adjustment of pH: No [actual pH: 7.62-7.81 (fresh media); 7.49-7.70 (expired media)]
- Photoperiod: 16 h light / 8 h darkness
- Light intensity: 540 – 1000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and visible abnormality (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc). Observations were made after 2 ± 0.5 h, 5 ± 1 h after the start of the exposure. On days 1-4 of the test, all vessels with living fish were inspected twice per day.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable. See below.
- Justification for using less concentrations than requested by guideline: 0 (control), and 100 mg/L nominal ; in the definitive: a saturated solution prepared with of 100 mg/L of the test item was tested in a limit test. Equivalent Geometric mean measured concentrations were determined: 0 (control) and 2.14 μg/L, based on analysis during the definitive test period. The limit concentration was selected based upon the very low solubility of the test item and in accordance with the OECD TG 203 Guidelines for testing. Additionally, the selection of the test concentration is based on the derivation of a threshold concentration (TC) from the data of the test item of results of a previous daphnia acute immobilization test (EC50 (0-48 h) = > saturated solution (nominal concentration of 100 mg/L) and of a previous alga toxicity test (ErC50 (0-72 h) = > saturated solution (nominal concentration of 100 mg/L). Related study citations provided in the full study report.
- Range finding study
- Test concentrations: Not applicable.
- Results used to determine the conditions for the definitive study: Not applicable.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 2.14 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% C.I.: - µg/L ; based on geometric mean measured concentrations ; > 100 mg/L nominal concentration

Remarks:

i.e. LC50 is above the solubility limit ; no mortality (and/or abnormal observations) below solubility limit

Details on results:

- Behavioural abnormalities: None. See tables.
- Observations on body length and weight: Not reported
- Other biological observations: None reported
- Mortality of control: 0%
- Other adverse effects control: No abnormalities observed in the control.
- Abnormal responses: None reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. Prior to start of the exposure intervals, the test solutions were checked for undissolved test item via laser beam (Tyndall effect). The saturated solution was visually clear. Presence of undissolved test item during preparation and during the test was not observed.
- Effect concentrations exceeding solubility of substance in test medium: Yes. The concentration of the test item was analytically verified via GC-MS/MS in fresh media at the start of the exposure interval (0 hours and 72 hours) and in the corresponding old media at the end of the exposure interval (24 hours and 96 hours) in the limit test item concentration and the control. The test item is known to break down (-lyse) under use conditions, meaning that in the aqueous conditions of this study, it is expected to be present as a mixture of the parent compound (test item) and the released components. In order to mimic those conditions, a saturated solution was prepared. The saturated solution was tested with a nominal loading rate of 100 mg/L of the test item in a limit test. The measured concentration of the test item (as a parent) was 0.163 μg/L at start and 0.110 μg/L at end of the first exposure interval and 41.1 μg/L at start and 28.6 μg/L at end of the fourth exposure interval, corresponding to a geometric mean measured concentration of 2.14 μg/L. No mortality or other adverse effects were observed in any treatment group at up to the limit of solubility or within the control group.
- Other: For the limit test item loading rate concentration (100 mg/L nominal) : the measured concentration of the test item was 0.163 μg/L at the test start (0 hours), 0.110 μg/L in old media (24 hours), 41.1 μg/L in fresh media (72 hours) at renewal and 28.6 μg/L in old media at test end (96 hours). The control concentrations were < LOQ at all time points. Equivalent Geometric mean measured concentrations were determined: 0 (control) and 2.14 μg/L, which were based on analysis during the definitive test period. The analytical values indicate that the sampling points were not completely homogeneous, which can be caused by the low water solubility of the test item, which is in accordance with the results of related studies (Water Solubility and/or Acute Daphnia Toxicity – cited in the full study report). Test item did not need to be corrected for recoveries as procedural recoveries (% recovery calculated to weighed amount of the test item) were within the range of 90% to 109% and with a mean 102%, during exposure period.

Sublethal observations / clinical signs:

Table 1. Nominal loading and measured concentrations of the test item during the exposure

Sampling date	Fresh Media, 0 hours	Old Media, 24 hours
Nominal loading of the test item [mg/L]	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%
	test item (as a parent)
100	0.163	0.110 #1)	76
Control	< LOQ	< LOQ
PC	93%	109% *)	-
Sampling date	Fresh Media, 72 hours	Old Media, 96 hours		Geometric mean measured test item concentration
	test item (as a parent)
Nominal loading of the test item [mg/L]	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%	[µg/L]
100	41.1 #2)	28.6	70	2.14
Control	< LOQ	< LOQ		< LOQ
PC	104%	90%	-

Meas. conc. = measured concentration of the test item, enrichment and dilution factors taken into account

% = percent of the initially measured concentration of the test item

#1) = mean value of four replicates

#2) = mean value of three replicates

LOQ = limit of quantification of the analytical method (0.025 μg/L of the test item).

PC = Procedural recovery, % recovery calculated to weighed amount of the test item.

*) Mean value of three replicates due to reanalyses

 

Table 2: Cumulative Mortality [%] in the Test Vessels

Nominal test item concentration [mg/L]	Cumulative mortality [%] at observation time [day/hours]
	day 0		day 1		day 2		day 3		day 4
	2	4	21	28	45	51	69	76	92	96
100	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Table 3: Observations of Sub-Lethal Effects and Normal Behaviour in the Test Vessels

(n = 7, number of fish)

Nominal test item concentration [mg/L]	Effect *	Number of fish affected at approximate observation time from start [day/hours]
		day 0		Day 1		day 2		day 3		day 4
		2	4	21	28	45	51	69	76	92	96
100	(1)	7	7	7	7	7	7	7	7	7	7
Control	(1)	7	7	7	7	7	7	7	7	7	7

*) : The numbers in brackets correspond to the following observations: 

(1) = Normal behaviour

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 for the test item to Danio rerio was determined to be > 2.14 (C.I: – ) μg/L based on geometric mean measured concentrations. No mortality and no adverse effects were observed in the saturated solution of test item (i.e. the 96h-LC50 was found to be greater than the limit of solubility of the test item).

Executive summary:

The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour semi-static test according to OECD TG 203 guideline under GLP. The test item is known to break down under use conditions, meaning that in the aqueous conditions of this study, it is expected to be present as a mixture of the test item parent compound and the released components. In order to mimic those conditions, a saturated solution was prepared. The saturated solution was tested with a nominal loading rate of 100 mg/L of the test item in a limit test. The concentrations were based on the derivation of a threshold concentration (TC) from the preceding data of the test item of results of an acute daphnia immobilization test (48h-EC50 > saturated solution or > 100 mg/L nominal) and/or an alga toxicity test (72h-ErC50 > saturated solution or > 100 mg/L nominal). The study was performed over a period of 96 hours under semi -static conditions with renewal of test solutions after every 24 hours as a worst-case exposure. Seven test organisms were exposed to the test concentration and the control, respectively. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The test solutions were visually clear throughout the study. The concentration of the test item was analytically verified via GC-MS/MS in fresh media at the start of the exposure interval (0 hours and 72 hours) and in the corresponding old media at the end of the exposure interval (24 hours and 96 hours) in the limit test item concentration and the control. The test item is designed to break apart under use conditions and therefore to be present in the test vessel as a mixture. The loading rate of the test item in this limit test was 100 mg/L and the measured concentration of the test item (as a parent) was 0.163 μg/L at start and 0.110 μg/L at end of the first exposure interval and 41.1 μg/L at start and 28.6 μg/L at end of the fourth exposure interval, corresponding to a geometric mean measured concentration of 2.14 μg/L.Test item did not need to be corrected for recoveries as procedural recoveries (% recovery calculated to weighed amount of the test item) were within the range of 90% to 109% and with a mean 102%, during exposure period. Air saturation was94-100% ASV (fresh media); 81-90% (expired media) and mortality in the control was less than 10% at the conclusion of the test. All validity criteria were considered to be met. Under the conditions of this study, the 96h LC50 was > 2.14 μg/L based on geometric mean measured concentrations or > 100 mg/L based on nominal loading rate of the test item. No mortality and no adverse effects were observed in the saturated solution of test item (i.e. the 96h-LC50 was found to be greater than the limit of solubility of the test item).

Description of key information

LC50 (fish) = > 2.14 µg/L based on geometric mean measured concentrations or > 100 mg/L based on nominal loading concentration, the 96h-LC50 was found to be greater than the limit of solubility of the test item, 96-hour, freshwater, OECD TG 203, 2020

 

Conclusion : The substance has apparent lower solubility in fish medium equivalent or similar to OECD TG 203 annex 3 (2019), than in pure water within as measured in the flask method to EU Method A.6 (2020). This is potentially a result of differences in ionic strength and other parameters between the aquatic exposure matrix and that of pure water. The conclusion of the experimental study was that no mortality and no adverse effects were observed at up to 100% saturation, with geometric mean measured concentration of 2.14 µg/L. Consequently, it can be concluded the test item has no toxicity below the water solubility limit observed experimentally.

Additional information

Key study : OECD TG 203, 2020 : The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour semi-static test according to OECD TG 203 guideline under GLP. The test item is known to break down under use conditions, meaning that in the aqueous conditions of this study, it is expected to be present as a mixture of the test item parent compound and the released components. In order to mimic those conditions, a saturated solution was prepared. The saturated solution was tested with a nominal loading rate of 100 mg/L of the test item in a limit test. The concentrations were based on the derivation of a threshold concentration (TC) from the preceding data of the test item of results of an acute daphnia immobilization test (48h-EC50 > saturated solution or > 100 mg/L nominal) and/or an alga toxicity test (72h-ErC50 > saturated solution or > 100 mg/L nominal). The study was performed over a period of 96 hours under semi -static conditions with renewal of test solutions after every 24 hours as a worst-case exposure. Seven test organisms were exposed to the test concentration and the control, respectively. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The test solutions were visually clear throughout the study. The concentration of the test item was analytically verified via GC-MS/MS in fresh media at the start of the exposure interval (0 hours and 72 hours) and in the corresponding old media at the end of the exposure interval (24 hours and 96 hours) in the limit test item concentration and the control. The test item is designed to break apart under use conditions and therefore to be present in the test vessel as a mixture. The loading rate of the test item in this limit test was 100 mg/L and the measured concentration of the test item (as a parent) was 0.163 μg/L at start and 0.110 μg/L at end of the first exposure interval and 41.1 μg/L at start and 28.6 μg/L at end of the fourth exposure interval, corresponding to a geometric mean measured concentration of 2.14 μg/L.Test item did not need to be corrected for recoveries as procedural recoveries (% recovery calculated to weighed amount of the test item) were within the range of 90% to 109% and with a mean 102%, during exposure period. Air saturation was 94-100% ASV (fresh media); 81-90% (expired media) and mortality in the control was less than 10% at the conclusion of the test. All validity criteria were considered to be met. Under the conditions of this study, the 96h LC50 was > 2.14 μg/L based on geometric mean measured concentrations or > 100 mg/L based on nominal loading rate of the test item. No mortality and no adverse effects were observed in the saturated solution of test item (i.e. the 96h-LC50 was found to be greater than the limit of solubility of the test item).