Research Article |
Corresponding author: Damjana Drobne ( damjana.drobne@bf.uni-lj.si ) Academic editor: Ingo S. Wehrtmann
© 2014 Damjana Drobne, Samo Drobne.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Drobne D, Drobne S (2014) Reference values for feeding parameters of isopods (Porcellio scaber, Isopoda, Crustacea). In: Wehrtmann IS, Bauer RT (Eds) Proceedings of the Summer Meeting of the Crustacean Society and the Latin American Association of Carcinology, Costa Rica, July 2013. ZooKeys 457: 313-322. https://doi.org/10.3897/zookeys.457.6805
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The advantage of using terrestrial isopods in toxicity studies is that a battery of parameters can be tested at different levels of biological complexity. Feeding parameters for example link organism level response to potential ecological consequences but a problem with using feeding parameters in toxicity tests with terrestrial isopods is their high variability. The aim of our study was to set benchmark values for feeding parameters for isopod Porcellio scaber (Isopoda, Crustacea) in laboratory-controlled experiments. In the work presented here, the daily feeding rate of the central 50% of the control population of P. scaber and a correlation between feeding rate and isopod weight were set. Values outside these ranges need additional evaluation to increase the relevance of test outcomes. We suggest using benchmark values for feeding parameters as well as the coefficient of variation (a) to identify animals with altered feeding parameters with respect to controls, and (b) to assess the data quality in each experiment.
Feeding behaviour, toxicity testing, standard tests
Numerous studies have analysed feeding behaviour and food preferences of terrestrial isopods, which have an important ecological role as decomposers of leaf litter and are one of the model organisms used in laboratory toxicity testing and terrestrial ecotoxicology (
Terrestrial isopods are saprophagous animals and have no single food source that meets all their nutritional needs. Only a mixture of several food items is an optimal nourishment (
Adaptations in feeding behaviour have allowed terrestrial isopods to reduce food consumption or be selective among different food items (
Since food consumption is indicative of the overall condition of an organism (
An intrinsic characteristic of feeding parameters is their relatively high variation. This fact is interpreted as “noise” when a desired signal is measured. As a result, the power of isopod toxicity tests with feeding rate as a measured response is reduced, and quantification of the response (signal) to toxicants is of limited power. In toxicity tests an adverse effect is proven when the signal-to-noise ratio equals or exceeds a certain value and when a clear dose-effect response is observed (
The aim of our study was to set the limits for feeding parameters of the terrestrial isopod Porcellio scaber (Isopoda, Crustacea) in laboratory-controlled experiments, which could serve as a benchmark for feeding rate of control, non-stressed animals. We analysed feeding parameters of about 600 animals in 60 experiments. In the analysed specimens, several sources of biological variability were retained to mimic to some degree the characteristics of isopod populations in nature. The sources of variability included: sex, moulting, different periods of laboratory acclimation, possible intracellular bacterial infection, and food quality. We discuss the interval for feeding rate, which could be taken as a characteristic for control animals – i.e. interquartile range – and suggest it as a benchmark for feeding rate of P. scaber in a laboratory single-species test.
Terrestrial isopods (Porcellio scaber, Isopoda, Crustacea) were collected between June 2006 and June 2013 at different locations in Slovenia, which were all considered to be unpolluted. Prior to experiments, the animals were kept in a terrarium filled with a layer of moistened soil and a thick layer of partly decomposed hazelnut tree leaves (Corylus avellana), at a temperature of 20 ± 2 °C and a 16:8-h light:dark photoperiod. They were acclimated to laboratory conditions for at least 14 days before the start of the experiment. Adult animals of both sexes, weighing more than 25 mg, were used in the experiments.
Hazelnut leaves were dried at room temperature, cut into pieces weighed and placed in a Petri dish. One animal was placed in each dish together with two to four pieces of leaves, which were the isopods only food source. Petri dishes were kept in a large glass container under controlled conditions in terms of humidity (≥80%), temperature (21 ± 1 °C) and light regime (16:8-h light:dark photoperiod). After 14 days, the leaf remnants were removed, dried and weighed.
Feeding rate per day (FR) was calculated as the amount of food consumed divided by the isopod fresh weight at the beginning of exposure (IW) per day.
The data presented here were obtained from 60 different experiments conducted with 594 animals between 2006 and 2013. These specimens served as control animals in different feeding experiments. All experiments were conducted following the same exposure protocol, and standard operational procedure (
where n is the number of observations, γ1 is the coefficient of skewness, and s is standard deviation of observed data. Correlation between observed variables was tested by a t-test. The interquartile range of feeding rate per day was chosen arbitrarily as a reference interval for the control animals. Interquartile range defines the central 50% of sample. The interquartile range of observed variables of control animals was estimated by confidence intervals for the first (Q1) and the third quartile (Q3) using:
where q is the quantile rank of Q1 (q=0.25) and Q3 (q=0.75) respectively, j (Q1) and j (Q3) were ranks of lower limits of the confidence interval of Q1 respectively Q3, k (Q1) and k (Q3) were ranks of upper limits of confidence interval of Q1 respectively Q3. Confidence intervals for quartiles were estimated for risk level at α < 0.05, α < 0.01 and α < 0.001. Analysis and graphical presentations were performed in SPSS 21 and Excel 2013.
Variation of feeding rate per day (FR), judging by the coefficients of variation, is twice the variation of the isopod weight (IW) at the beginning of the experiment (Table
Table
Descriptive statistics for animals (Porcellio scaber) from laboratory-feeding experiments.
Statistics | Isopod weight at the beginning of experiment (in mg; IW) |
Feeding rate per day (in mg/g; FR) |
---|---|---|
Number (n) | 594 | 594 |
Minimum (min) | 15.800 | 0.001 |
Maximum (max) | 105.200 | 0.257 |
Mean (m) | 44.800 | 0.055 |
Variance (s2) | 177.672 | 0.001 |
Standard deviation (s) | 13.329 | 0.033 |
Coefficient of variation (CV) | 0.298 | 0.599 |
Skewness (γ1) | 1.033 | 2.251 |
Kurtosis (γ2) | 1.879 | 7.916 |
First quartile (Q1) | 35.400 | 0.035 |
Median (Me) | 43.050 | 0.048 |
Third quartile (Q3) | 52.125 | 0.068 |
Isopod weight at the beginning of experiment (in mg; IW) |
Feeding rate per day (in mg/g; FR) |
---|---|
Pr[34.28 < Q1 < 36.40]=0.95 | Pr[0.033 < Q1 < 0.037]=0.95 |
Pr[50.93 < Q3 < 53.14]=0.95 | Pr[0.065 < Q3 < 0.071]=0.95 |
Pr[33.78 < Q1 < 36.80]=0.99 | Pr[0.032 < Q1 < 0.037]=0.99 |
Pr[50.43 < Q3 < 54.00]=0.99 | Pr[0.062 < Q3 < 0.073]=0.99 |
Pr[33.48 < Q1 < 37.01]=0.999 | Pr[0.032 < Q1 < 0.038]=0.999 |
Pr[49.83 < Q3 < 54.20]=0.999 | Pr[0.062 < Q3 < 0.074]=0.999 |
In the case where the probability Pr=0.99 was chosen, feeding rate per day (FR) of the central 50% of the control population was situated in the interval [0.032,0.073] mg/g; Pr[0.032< FR<0.073]=0.99. In addition, if probability was lower (Pr=0.95), the result for the interquartile range of feeding rate per day was similar (Pr[0.033< FR<0.071]=0.95). Therefore, the central 50% of values of feeding rate per day were in the interval [0.03< FR<0.07] mg/g.
The reference values for our laboratory control P. scaber was between 0.03 and 0.07 mg/g of feeding rate per day ([0.03< FR<0.07] mg/g) when their weight at the beginning of the test was in the range between 34 and 54 mg ([34< IW<54] mg) (Figs
Terrestrial isopods of the species Porcellio scaber are among the most frequently used species in (eco)toxicity testing, and feeding parameters are among more relevant ecotoxicity endpoints (
Benchmark values for measured parameters in control animals allow direct comparison of measurement results in proficiency testing (round robin testing) performed by different laboratories (
The data for feeding rate were not-normally distributed and nonparametric statistics must be used for further analysis. Not-normal data distribution can be explained by the fact that some animals could eat less or even stop eating, but tested animals could not eat above a certain limit. The coefficient of variation allowed us to compare the variation of different data. When we correlated feeding and isopod weight, it appeared that the correlation is relatively low suggesting that feeding behaviour of animals depends not only on weight but also on other parameters, which have not been tested yet or are even unknown. Our results suggest that data for feeding rate have higher variability as data for isopod weight.
Information on both technical and biological variance is of major importance in interpretation of the results of toxicity testing and in assessing their quality. Technical variance is attributed to performance of experiments and experimental conditions, while the biological source of variance is an inherited biological variability of the measured parameter. In our tests, most of the variance originated from the biological variability, and the technical variance was presumed to be low due to controlled and constant experimental conditions. Our data confirm that variance is an intrinsic characteristic of feeding parameters. Analysing and understanding the variance is of significant importance in the decision-making process used to set proper safety factors (
In a single species laboratory test with P. scaber several endpoints are commonly assessed. These are biochemical biomarkers, histopathological changes, behavioural responses and physiological alterations as well as different organism level responses including moulting, mortality and growth. The selected biomarkers vary in sensitivity, ease of observation, reproducibility, repeatability and ecological importance (
Feeding rate of terrestrial isopod P. scaber either decreases or increases due to food additives. The significance of this change is difficult to assess when a parameter has high background variability. We suggest using reference, benchmark data for feeding parameters and coefficients of variation for feeding rate to: (a) discriminate between animals with altered feeding parameters with respect to a control in addition to existing statistical tests; (b) assess the data quality in future experiments by comparing of the coefficient of variation and the established range for feeding parameters in control animals. In addition, establishing also reference, resting values for other parameters analysed in toxicity tests could increase the relevance of toxicity data such as ECx, LOEC, NOEC, and could assist significantly in quality control of the data.
We thank to PhD students Ziva Pipan Tkalec, Anita Jemec, Janez Valant, Sara Novak, Miha Golobic, Anja Menard, Tea Romih, Barbara Drasler, Veno Kononeko, Monika Kos, Alenka Malovrh and some others. Part of work was conducted within research projects financed by the Slovenian Research Agency (J1-9475, J1-4109) and within the seventh FP EU Project ‘‘NANOVALID’’ (Contract No. 263147). We thank G.W.A. Milne for editorial assistance.