Gum of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by extraction with alkaline solution

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:

2015-04-29 to 2016-02-23

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:

referenced as Method C.2 of Commission Regulation No. 440/2008

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Not applicable

Analytical monitoring:

yes

Details on sampling:

Chemical analyses: Single samples for analysis were taken from the control and all test solutions at the start of the test (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

Vehicle:

no

Details on test solutions:

The study was carried out using WAFs (Water Accommodated Fractions) since the test item was not well soluble in test water. The WAFs (for the fresh media at t=0h and t=24h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion.
The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the WAFs. The volume of the mixing vessel was approximately 1 L (except for the loading rate at 1 mg.L-1 with a 5-L bottle). A magnetic stirring bar was placed in each mixing vessel completely filled with test water (without headspace). The loading rates of the test item were weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were immediately sealed with screw caps after weighing. Each glass flask was placed in a water bath for 10-15 minutes at 50°C (following recommendations of the Sponsor), followed by sonication for ca. 10 minutes. This step was essential to remove the gum pellets stuck to the glass of the flasks (especially at the highest loading rates) and to extract a maximum of soluble fraction of the test item as possible. Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 +/- 2 hours of gentle stirring at room temperature, the WAFs were allowed to stand for 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs (at loading rates of 1.0 and 10 mg.L-1) were extracted via the drain port and directly added into test tubes (without headspace). Given that a non-negligible number of particles from the non-soluble fraction of the test item was observed at the bottom of the mixing vessel at the loading rate of 100 mg.L-1, the WAF was withdrawn from the middle of the bottle using a 10-mL glass pipette in order to avoid adding too much particles in test tubes. After filling and introduction of daphnids the test tubes were sealed immediately.
No small bubble was observed in the test tubes. The test solution was observed to be clear and colourless (at loading rates of 1.0 and 10 mg.L-1) to slightly yellow (only for the loading rate of 100 mg.L-1). The test was carried out without adjustment of the pH.

Based on the results of a range-finding test solutions used in the definitive test were prepared to obtain the following loading rates: 1.0, 10 and 100 mg.L-1.
Test water without test substance but treated in the same way as the test substance solutions (WAFs).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Age at Test Start: < 24 hours old
- Feeding during test: No feeding


ACCLIMATION
At least 48 hours prior to the start of the test, gravid daphnids were transferred to OECD test water and held at similar temperature and light conditions as used in the test. During this period, daphnids were fed in the same manner as that of the stock population. Only daphnids up to 24 hours old were used for the test.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Reconstituted water, as prescribed by the OECD Guideline 202

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

250 mg.L-1 (as CaCO3)

Test temperature:

20 °C ± 2 °C

pH:

6.0-9.0, not varying by more than 1.5 units

Dissolved oxygen:

Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

1.0, 10 and 100 mg test item.L-1 nominal loading rates.

Details on test conditions:

TEST SYSTEM
A semi-static test was performed with renewal of test solutions after 24 hours (because of the properties of the test item).
All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and concentration.
4 replicates with daphnids for the control and per loading rates.
Chemical analyses were taken from additional replicates in order to avoid disturbing the daphnids at t=24h and to prevent potential test item losses on opening test tubes:
- two additional series for sampling for analysis of test concentrations from the freshly prepared solutions (t=0h and t=24h fresh)
- and another for sampling from the 24-hour old solutions (t=24h old).

The sampling for analysis of the control and test concentrations at t=48h was performed directly from one of the 4 remaining replicates with daphnids after reading of the immobilisation test.
Number of Daphnids 20 per control and test concentration (loading rate), divided into 4 groups of 5 animals
Loading 5 daphnids per vessel each completely filled with test solution and without headspace

TEST MEDIUM / WATER PARAMETERS
Reconstituted water, as prescribed by the OECD Guideline 202.
Stock solutions: a) CaCl2.2H2O 11.76 g.L-1
b) MgSO4.7H2O 4.93 g.L-1
c) NaHCO3 2.59 g.L-1
d) KCl 0.23 g.L-1
An aliquot (25 mL) of each solution (a to d) was added to each litre (final volume) of deionised water (conductivity < 10 μS.cm-1).
The pH of this solution was in the range of 6 to 9 and the total water hardness was approximately 250 mg.L-1 (as CaCO3). This reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value, and then set aside for 2 days without aeration.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light : 8h dark
- Light intensity:

EFFECT PARAMETERS MEASURED
Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Immobile animals were eliminated from the vessels as soon as they were discovered. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.


RANGE-FINDING STUDY
- Test concentrations: loading rates of 1.0, 10, 50 and 100 mg.L-1
- Results used to determine the conditions for the definitive study: 10% of immobilisation after 48h at a loading rate of 100 mg test item/L-1.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rates of test item

Basis for effect:

mobility

Details on results:

After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the nominal test concentrations 1.0 and 10 mg.L-1 (loading). After 48 hours of exposure, only 10% immobilisation was observed at the nominal test loading rate of 100 mg.L-1. It should be noted that after 24 and 48 hours of exposure some residual particles were lying at the bottom of the test tubes at the highest test loading rate.
The presence of these particles was unavoidable since WAFs were not filtered in order to preserve a maximum of the composition of the WAF and to prevent potential losses of the test item due to filtration. These particles could be the cause of the observed immobilisations on daphnids (physical effect) rather than toxicity due to the test substance, but regarding results of COD content in WAFs an effect directly due to a test item effect is not excluded. However, this should be taken cautiously because the test item was probably in excess and thus some constituents may have been above their solubility limit.

Results with reference substance (positive control):

No data.

Reported statistics and error estimates:

No data.

Validity criteria

Controls: In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress.

Dissolved [02]: Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels. Thus the validity criteria have been fulfilled in the present study.

Water quality parameters and environmental conditions throughout the test

pH and oxygen values remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen: ≥ 60% of the air-saturation value at the end of the test). It should be noted that a slight decrease in pH values was observed in particular at 100 mg.L-1 in comparison with pH values of the control.

The temperature of the test medium recorded continuously in the vessel next to the test vessels was situated between 20.8 and 22.0 °C throughout the test (average value: 21.4 °C), which was slightly higher than the requirements as laid down in the study plan (20 °C ± 2 °C and constant within 1 °C). This minor deviation was considered not to affect the results of the test as no adverse effects of exposure were observed in the control throughout the duration of the test.

pH-Values during the Final Test

		Nominal Concentration (mg test item.L-1)*
		Control	1	10	100
Start t=0h		7.95	7.93	7.84	7.75
t=24h	Old	7.84	7.82	7.83	7.68
	Fresh	7.95	7.94	7.85	7.65
End t=48h		7.58	7.63	7.66	7.57

Dissolved Oxygen Concentrations (mg.L-1) during the Final Test

		Nominal Concentration (mg test item.L-1)*
		Control	1	10	100
Start t=0h		8.33	8.35	8.3	8.21
t=24h	Old	8.01	8.09	8.02	7.91
	Fresh	8.19	8.33	8.25	8.06
End t=48h		8.22	8.19	7.95	7.88**
* WAF prepared at the given loading rate. ** corresponding to a value > 88% of the air-saturation value

Chemical analysis & Analytical monitoring

Samples taken from test concentrations were analysed at the start, at t=24h (new and old solutions) and at the end of the test in order to check the presence of two key constituents (α-Pinene and Hydrocinnamic Acid) of the test item. Moreover, concentration of dissolved organic material in the WAFs was checked by analysis of the Chemical Oxygen Demand (ST-COD) in the control medium and the WAFs.

Although every effort was made in a first time to extract and solubilise the soluble fraction of the test item in test water (heating, sonication, mixing without headspace …) and secondly to maintain the concentrations of the WAFs (semi-static and closed conditions without headspace), COD analyses indicate that unquantifiable organic compounds were found in WAFs, except at the loading rate of 100 mg.L-1.

Measured Concentrations of the Test Item in Test Water - Results of the Determination of the COD Analysis

		COD (mg O2.L-1)
		Control	1	10	100
Start t=0h		< LOQ	< LOQ	< LOQ	8
t=24h	Old	< LOQ	< LOQ	< LOQ	10
	Fresh	< LOQ	< LOQ	< LOQ	17
End t=48h		< LOQ	< LOQ	< LOQ	9

Levels of α-Pinene were below the LOQ and the LOD. Analytical monitoring of Hydrocinnamic Acid showed that concentrations of this last constituent at loading rates of 10 and 100 mg.L-1 were

satisfactorily maintained within ± 20% of the initial concentration throughout the test, except the second day at the nominal test loading rate of 10 mg.L-1 with losses ca. 37%. It should be noted that chemical analyses were performed only to provide an indication of the concentration of dissolved organic material in the WAFs and their stability. A WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation…). Due to these differences, the ratio of certain constituents may change over time and the composition of the WAFs after 24h will not be exactly that of the fresh solutions. Moreover, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the same way that if it was alone in the mixture.

Thus, since the study was carried out using WAFs of a natural complex substance made of several constituents with different stability and behaviours in aqueous solutions during testing, it was considered acceptable to base results on nominal loading values.

Measured concentrations (mg.L-1) of the test substance in test water – Final test. Hydrocinnamic Acid.

Nominal concentration* (mg test item.L-1)	Start (t=0h)	t=24h Old	Relative loss to initial value (%)	t=24h Fresh	End (t=48h)	Relative loss to initial value (%)
Control	Absence	Absence	N.A.	Absence	Absence	N.A.
1	Absence	Absence	N.A.	Presence	Absence	N.A.
10	0.1	0.1	3	0.18	0.11	37
100	0.49	0.48	1	0.85	0.76	10

Measured concentrations (mg.L-1) of the test substance in test water – Final test. α-Pinene.

Nominal concentration* (mg test item.L-1)	Start (t=0h)	t=24h Old	Relative loss to initial value (%)	t=24h Fresh	End (t=48h)	Relative loss to initial value (%)
Control	Absence	Absence	N.A.	Absence	Absence	N.A.
1	Absence	Absence	N.A.	Absence	Absence	N.A.
10	Absence	Absence	N.A.	Absence	Absence	N.A.
100	Absence	Absence	N.A.	Absence	Absence	N.A.

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal loading rates of the test item, the 48-hour EL50 value was determined to be higher than 100 mg test item.L-1.

Executive summary:

A study was performed to assess the acute toxicity of the test item LABDANUM GUM to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). The criterion measured was the EL50 (Median Effective Loading rate), a statistically derived loading rate which is expected to cause immobility in 50% of test animals within a period of 48 hours.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rate of 1.0, 10 and 100 mg test item.L-1 and to a control. The immobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the WAFs was checked by analysis of the Chemical Oxygen Demand (COD) in the control medium and the WAFs at the start, at t=24h (new and old solutions) and at the end of the test. Analytical monitoring of the two constituents (α-Pinene and Hydrocinnamic Acid) was also performed.

Although every effort was made in a first time to extract and solubilise the soluble fraction of the test item in test water (heating, sonication, mixing without headspace …) and secondly to maintain the concentrations of the WAFs (semi-static and closed conditions without headspace), COD analyses indicate that unquantifiable organic compounds were found in WAFs, except at the loading rate of 100 mg.L-1. Levels of α-Pinene were below the LOQ and the LOD. Analytical monitoring of Hydrocinnamic Acid showed concentrations satisfactorily maintained within ± 20% of the initial concentration throughout the test at loading rates of 10 and 100 mg.L-1, except the second day at the nominal test loading rate of 10 mg.L-1 with losses ca. 37%.

After 48 hours of exposure, no immobilisation of the test animals was observed in the control and the nominal test loading rates of 1.0 and 10 mg.L-1. Only 10% immobilisation was observed at the nominal test loading rate of 100 mg.L-1 but this was probably not a substance related effect. Indeed, some residual particles of the non-soluble fraction of the test item were lying at the bottom of the test tubes after 24 and 48 hours of exposure at the highest test loading rate. The presence of these particles was unavoidable since WAFs were not filtered in order to preserve a maximum of the composition of the WAF and to prevent potential losses of the test item due to filtration. These particles could be the cause of the observed immobilisations on daphnids (physical effect) rather than toxicity due to the test substance.

Based on these results, the 24 and 48-hour EL50 were therefore > 100 mg test item.L-1.

This study respected the requirements of the guideline and the validity criteria have been fulfilled. Therefore, the present study is considered acceptable for that endpoint.

Results synopsis:

Test: Toxic effect of LABDANUM GUM to Daphnia magna according to OECD guidelines No. 202.

48h-EL50: > 100 mg/L

Description of key information

Under the experimental conditions and based on nominal loading rates of the test item, the 48-hour EL50 value was determined to be higher than 100 mg test item/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

For that endpoint, a study on the registered substance was available. In this study, the toxic effect of the test item LABDANUM GUM to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). The criterion measured was the EL50, a statistically derived loading rate which is expected to cause immobility in 50% of test animals within a period of 48 hours.

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rate of 1.0, 10 and 100 mg test item.L-1 and to a control. The immobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the WAFs was checked by analysis of the Chemical Oxygen Demand (COD) in the control medium and the WAFs at the start, at t=24h (new and old solutions) and at the end of the test. Analytical monitoring of the two constituents (α-Pinene and Hydrocinnamic Acid) was also performed.

Although every effort was made in a first time to extract and solubilise the soluble fraction of the test item in test water (heating, sonication, mixing without headspace …) and secondly to maintain the concentrations of the WAFs (semi-static and closed conditions without headspace), COD analyses indicate that unquantifiable organic compounds were found in WAFs, except at the loading rate of 100 mg.L-1. Levels of α-Pinene were below the LOQ and the LOD. Analytical monitoring of Hydrocinnamic Acid showed concentrations satisfactorily maintained within ± 20% of the initial concentration throughout the test at loading rates of 10 and 100 mg.L-1, except the second day at the nominal test loading rate of 10 mg.L-1 with losses ca. 37%.

After 48 hours of exposure, no immobilisation of the test animals was observed in the control and the nominal test loading rates of 1.0 and 10 mg.L-1. Only 10% immobilisation was observed at the nominal test loading rate of 100 mg.L-1 but this was probably not a substance related effect. Indeed, some residual particles of the non-soluble fraction of the test item were lying at the bottom of the test tubes after 24 and 48 hours of exposure at the highest test loading rate. The presence of these particles was unavoidable since WAFs were not filtered in order to preserve a maximum of the composition of the WAF and to prevent potential losses of the test item due to filtration. These particles could be the cause of the observed immobilisations on daphnids (physical effect) rather than toxicity due to the test substance. Therefore, under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value was found to be higher than 100 mg test item.L-1.

Despite some minor deviations that did not affect the integrity of the study, the study respected the requirements of the guideline and the validity criteria were successful. This study was therefore considered acceptable for that endpoint.